Well plate incubator

ABSTRACT

Incubators including an enclosure with an internal chamber configured to support a cell culture plate comprising a plurality of wells are disclosed. The enclosure includes a plurality of openings configured to allow access to the wells. The incubators include a sealing element configured to seal the plurality of openings in the enclosure. The sealing element comprises a plurality of openings corresponding to at least a subset of the plurality of openings in the enclosure. Access to the internal chamber can be provided by aligning the plurality of openings in the sealing element with the plurality of openings in the enclosure. Methods for using the incubators are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119 of U.S. PatentApplication No. 62/235,863 titled “Well-Plate Incubator” filed on Oct.1, 2015, the disclosure of which is incorporated herein by reference.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

BACKGROUND

Incubators can be used to hold samples containing materials, includingmicro-objects and other components derived from biological cells, andprovide conditions to maintain the viability of biologically relatedmaterials. For example, the interior environment of the incubator canhave a certain temperature range, humidity, and carbon dioxide contentselected to maintain the viability of the materials.

The materials maintained within the incubator can be accessed by openingthe incubator. However, opening the incubator, such as by opening a lidof the incubator, can introduce contaminants and disrupt the interiorenvironment of the incubator. Repeated opening can adversely affect thebiological viability of the materials within the incubator.

Accessing the interior of the incubator with a robotic arm can also bedifficult to automate because of the significant complexity of movementrequired by the robotic arm in order to open and access the interior ofthe incubator. Even if the robotic arm is configured to access theincubator after opening the lid, the extra steps can significantlydecrease process throughput. Repeated opening of the lid in combinationwith the use of a robotic arm can adversely affect the materials. Onesolution that has been developed to address this problem is to locatethe robotic arm and incubator within a larger incubator having aninternal environment with conditions selected to maintain viability ofthe materials. However, this solution creates additional problems forthe equipment operating within the incubator environment. For example,the tooling and equipment maintained in the environment is subject toadditional condensation that can damage or inhibit the robotic arm.Enlarging the incubator environment also greatly increases thecomplexity and costs of the system.

A need therefore exists for an incubator that addresses many of theseissues and that can be easily accessed by a robotic arm or otherimport/export tip while maintaining the internal incubator environmentto support the viability of biological and other materials.

SUMMARY OF THE DISCLOSURE

The present invention relates to incubators having a plurality ofopenings that can provide access to wells in a cell culture platesupported within the incubator. The incubators can improved access whilepreventing contamination of the environment within the incubator.

In one aspect of the invention, an incubator is provided, where theincubator includes an enclosure having an internal chamber configured tosupport a cell culture plate comprising a plurality of wells, theenclosure including a plurality of openings configured to allow accessto the wells of the cell culture plate; and a sealing element configuredto seal the plurality of openings in the enclosure, the sealing elementincluding a first plurality of openings corresponding to at least asubset of the plurality of openings in the enclosure.

In some embodiments of the incubator, each opening of the plurality ofopenings in the enclosure may have a diameter of about 1 mm to about 10mm. In some other embodiments, each opening of the plurality of openingsin the enclosure may have a diameter of about 1 mm to about 5 mm, orabout 1.0 mm, about 1.5 mm, about 2.0 mm, about 2.5 mm, about 3.0 mm,about 3.5 mm, about 4.0 mm, about 4.5 mm, or about 5.0 mm, or any rangedefined by one of the foregoing sizes.

In various embodiments of the incubator, the internal chamber of theenclosure may have a volume of about 50 cm³ to about 300 cm³. In otherembodiments, the internal chamber may have a volume of about 100 cm³ toabout 500 cm³. In yet other embodiments, the internal chamber may have avolume of about 200 cm³ to about 750 cm³. Alternatively, the internalchamber may have a volume of about 400 cm³ to about 1,000 cm³. Infurther embodiments, the internal chamber may have a volume of about 500cm³ to about 1500 cm³. In other embodiments, the internal chamber mayhave a volume of about 750 cm³ to about 2000 cm³.

In various embodiments of the incubator, the cell culture plate may be a96-well plate. In other embodiments, the cell culture plate may be a384-well plate. In some other embodiments, the cell culture plate mayhave 24 or fewer wells (e.g., 12 wells, 6 wells, etc.).

In various embodiments of the incubator, the enclosure may include abase and a lid, the base and the lid defining the internal chamber. Inother embodiments, the enclosure may include a base, a lid, and a frontplate, the base, the lid, and the front plate defining the internalchamber. The base may be formed from a rigid material having a highthermal conductivity and low thermal capacitance. In some embodiments,the base may be configured with a hollow region forming part or all ofthe internal chamber of the enclosure. In some embodiments, the base mayinclude a bottom and four walls with one of the four walls having aheight that is shorter than the height of the other three walls. Invarious embodiments, the lid is formed from an insulating plastic. Insome embodiments, the lid may include an outer surface (e.g., a surfacethat interfaces with air located outside of the incubator) and an innersurface within the enclosure (e.g., a surface that interfaces with airlocated within the inner chamber of the enclosure). The inner surface ofthe lid may include one or more recesses. In some embodiments, the lidmay include one or more connectors configured to sealably connect thelid to the base. In some embodiments, the one or more connectors mayinclude a magnet, a tab (e.g., a flexible tab), and/or a clip.

In various embodiments of the incubator, the plurality of openings inthe enclosure may be configured to be in register with the plurality ofwells in the cell culture plate. In some embodiments, access to theinternal chamber of the enclosure, and any cell culture plate containedtherein, may be provided by positioning the sealing element such thatone or more of the plurality of openings in the enclosure is in registerwith one or more openings in the sealing element. In variousembodiments, the sealing element may be movable between a closedposition, in which the sealing element occludes each of the plurality ofopenings in the enclosure, and a first open position, in which the firstplurality of openings of the sealing element are in register with the atleast a subset of the plurality of openings in the enclosure. In someembodiments, the number of openings in the first plurality of openingsof the sealing element may be the same as the number of openings in theenclosure. In other embodiments, the number of openings in the firstplurality of openings of the sealing element may be less than the numberof openings in the enclosure.

In some embodiments, the sealing element may further include a secondplurality of openings, the second plurality of openings being differentfrom the first plurality of openings. In some embodiments, the number ofopenings in the first plurality of openings and/or the second pluralityof openings in the sealing element is less than the number of openingsin the enclosure. In various embodiments, the sealing element mayfurther include a third plurality of openings, the third plurality ofopenings being different from the first plurality of openings and thesecond plurality of openings. In some of the embodiments, the number ofopenings in the first plurality of openings, the second plurality ofopenings, and/or the third plurality of openings in the sealing elementmay be less than the number of openings in the enclosure. For example,the number of openings in each of the first plurality of openings, thesecond plurality of openings, and the third plurality of openings in thesealing element can be less than the number of openings in theenclosure, while the sum of the first, second, and third pluralities ofopenings in the sealing element can be equal to the number of openingsin the enclosure. In some embodiments, the number of openings in thesecond plurality of openings in the sealing element may be one-half,one-third, or one-fourth the number of openings in the enclosure. Insome embodiments, the number of openings in the third plurality ofopenings in the sealing element may be one-third or one-fourth thenumber of openings in the enclosure.

In some embodiments, each of the plurality of openings in the sealingelement may have a diameter of about 1 mm to about 10 mm. In otherembodiments, each of the plurality of openings in the sealing elementhas a diameter of about 1 mm to about 5 mm, or about 1.0 mm, about 1.5mm, about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0mm, about 4.5 mm, about 5.0 mm, or any range defined by one of theforegoing sizes.

In various embodiments of the incubator, the sealing element may belocated inside the internal chamber of the enclosure. In variousembodiments, the sealing element may be movable between a closedposition and a first open position, wherein, when the sealing element isin the closed position, each of the plurality of openings in theenclosure may be occluded, and when the sealing element is in the firstopen position, a first plurality of openings in the sealing element maybe in register with a first subset of the plurality of opening in theenclosure and all other openings (if any) of the plurality of openingsin the enclosure may be occluded. In related embodiments, the sealingelement may be further movable to a second open position, wherein, whenthe sealing element is in the second open position, a second pluralityof openings in the sealing element (which may be identical to ordifferent than the first plurality of openings in the sealing element)may be in register with a second subset of openings in the enclosure andall other openings of the plurality of openings in the enclosure may beoccluded. In some embodiments, the first subset of openings in theenclosure and the second subset of openings in the enclosure may benon-overlapping subsets. In other related embodiments, the sealingelement may be further movable to a third open position, wherein whenthe sealing element is in the third open position, a third plurality ofopenings in the sealing element (which may be identical to or differentthan the first plurality and/or second plurality of openings in thesealing element) may be in register with a third subset of openings inthe enclosure and all other openings of the plurality of openings in theenclosure may be occluded. In some embodiments, the first, second, andthird subsets of openings in the enclosure may be non-overlappingsubsets. In some embodiments, the first, second, and third subsets ofopenings in the enclosure may be overlapping subsets (e.g., partiallyoverlapping).

In various embodiments of the incubator, the incubator may furtherinclude a sealing element actuator configured to move the sealingelement between a first open position and a closed position. In someembodiments, the sealing element actuator may be configured to move thesealing element between a second open position and the closed position.In some embodiments, the sealing element actuator may be configured tomove the sealing element between a third open position and the closedposition. In some embodiments, moving the sealing element to the firstopen position may include aligning openings of the sealing element(e.g., a first plurality of openings) with a first subset of theplurality of openings in the enclosure. In some embodiments, moving thesealing element to the second open position may include aligningopenings of the sealing element (e.g., the first plurality of openingsor a second plurality of openings) with a second subset of the pluralityof openings in the enclosure. In some embodiments, moving the sealingelement to the third open position may include aligning openings of thesealing element (e.g., the first plurality of openings, the secondplurality of openings, or a third plurality of openings) with a thirdsubset of the plurality of openings in the enclosure. In someembodiments, the sealing element actuator may include a motor or rotarysolenoid.

In various embodiments of the incubator, the incubator may furtherinclude at least one passage in the enclosure configured for gas entry.In some embodiments, the at least one passage configured for gas entrymay be located on a wall of the base, at the same height from a bottomof the base as a side of a cell culture plate held by a support withinthe inner chamber of the enclosure. In various embodiments of theincubator, the incubator may further include a connector adapted toconnect a pressurized gas source to a passage in the enclosureconfigured for gas entry. In related embodiments, the sealing elementmay be configured to form a seal with the plurality of openings in theenclosure that allows the enclosure to maintain a pressure in theinternal chamber between about 0.0005 psi to about 0.01000 psi aboveambient pressure when gas from the pressurized gas source flows into theinternal chamber. In various embodiments of the incubator, the incubatormay include at least one fluid drain passage in the enclosure configuredto drain a fluid reservoir within the enclosure. In some embodiments,the fluid drain passageway may be sealable.

In various embodiments of the incubator, the incubator may furtherinclude a printed circuit board (PCB). In some embodiments, the PCB islocated proximal to an internal surface of a top (e.g., a lid) of theenclosure. In various embodiments, the PCB includes a plurality ofopenings in register with the plurality of openings passing through theenclosure. For example, the PCB openings can be in register with aplurality of openings passing through a lid of the enclosure. In someembodiments, the PCB is located immediately adjacent to the sealingelement of the incubator. For example, the PCB can have a substantiallyflat surface that directly contacts a substantially flat surface of thesealing element. In certain embodiments, the sealing element is disposedbetween the PCB and an internal surface of a lid of the enclosure. Invarious embodiments, the incubator may further include one or moresensors on the PCB. In some embodiments, each of the one or more sensorsis selected from the group consisting of: a temperature sensor, ahumidity sensor, an oxygen sensor, and a carbon dioxide sensor.

In various embodiments of the incubator, the incubator may furtherinclude a temperature controller configured to maintain a temperature ofthe internal chamber within a desired range.

In various embodiments of the incubator, the incubator may furtherinclude a first heating/cooling device engaged with or otherwise coupledwith the enclosure, the first heating/cooling device controlled by thetemperature controller. In some embodiments, the first heating/coolingdevice may be selected from the group consisting of: a resistive heater,a fluid coil configured to circulate a heat exchange fluid, and one ormore Peltier devices. In some embodiments, the first heating/coolingdevice may directly or indirectly contact an outer surface of the bottomof the enclosure. In some embodiments, the first heating/cooling devicemay contact (directly or indirectly) at least about 75% of the outersurface of the bottom of the enclosure. In some embodiments, the firstheating/cooling device may include a fluid coil.

In various embodiments of the incubator, the incubator may furtherinclude a second heating/cooling device engaged with or otherwisecoupled with the enclosure, the first heating/cooling device controlledby the temperature controller. In some embodiments, the secondheating/cooling device may be engaged with a top (e.g., a lid) of theenclosure. In some embodiments, the second heating/cooling device may belocated within the enclosure. In some embodiments, the secondheating/cooling device may include a plurality of openings that are inregister with the plurality of openings in the enclosure. In someembodiments, the second heating/cooling device may include resistiveheating elements that are part of a PCB (e.g., the PCB described aboveand elsewhere herein).

In some embodiments, the resistive heating elements may be located on aside of the PCB facing the internal chamber of the enclosure. In otherembodiments, the resistive heating elements may be located within thePCB. For example, the PCB may comprise a multi-layer (e.g., four-layer)construction and the resistive heating elements may reside in internallayers of the PCB.

In various embodiments of the incubator, the incubator may furtherinclude a spacer having a plurality of openings. In some embodiments,the plurality of openings on the spacer may be in register with theplurality of openings of the enclosure. In some embodiments, the spacermay be located between the PCB and the sealing element. In otherembodiments, the spacer may be located between the sealing element andan internal surface of a lid of the enclosure. In some embodiments, thespacer may be configured to reduce friction between the sealing elementand the PCB or the internal surface of the lid of the enclosure when thesealing element moves between an open and a closed position. In someembodiments, the spacer may be configured to improve the seal formedbetween the sealing element and the PCB or the internal surface of thelid of the enclosure when the sealing element moves between an open anda closed position.

In various embodiments of the incubator, the incubator may furtherinclude a support for the cell culture plate. In some embodiments, thesupport may be configured to slideably move relative to the enclosurefrom a position within the enclosure to a position outside of theinternal chamber of the enclosure. In some embodiments, the support maybe formed by one or more internal surfaces of the enclosure.

In various embodiments of the incubator, the incubator may furtherinclude an access door attached to the support for the cell cultureplate. In some embodiments, the support and access door may form anaccess assembly including a front plate that sealably interfaces with aportion of the enclosure. In some embodiments, the access assembly maybe movably mounted on an enclosure support that supports the enclosure.

In various embodiments of the incubator, the incubator may furtherinclude tracks on the enclosure support, wherein the access assembly isconfigured to slide relative to the tracks on the enclosure support.

In various embodiments of the incubator, the incubator may furtherinclude an enclosure support configured to support the enclosure. Invarious embodiments of the incubator, the incubator may further includeone or more adjustable connectors configured to connect the enclosuresupport to the enclosure.

In various embodiments of the incubator, the incubator may furtherinclude an insulation material coupled to the enclosure. In someembodiments, the insulation material may be attached to one or moreouter surfaces of the enclosure. In various embodiments, the incubatormay be configured to maintain a selected internal temperature, humidity,and gas content within the internal chamber of the enclosure. In variousembodiments of the incubator, the incubator may further include acontroller configured to maintain the selected internal temperature,humidity, and gas content within the internal chamber of the enclosure.

In another aspect, the invention provides a method for accessing aninternal chamber of an incubator. The incubator can be any incubatordescribed above or elsewhere herein. For example, the incubator cancomprise an enclosure having a plurality of openings and a sealingelement having a plurality of openings corresponding to at least asubset of the plurality of openings in the enclosure. In variousembodiments, the method includes the steps of moving the sealing elementto an open position to bring the plurality of openings in the sealingelement into register with a first subset of openings of the pluralityof openings in the enclosure, the plurality of openings in the sealingelement and the first subset of the plurality of openings in theenclosure thereby providing a plurality of passages from an exterior ofthe incubator to the internal chamber of the enclosure; advancing animport/export tip through one or more of the plurality of passagesbetween the exterior of the incubator and the internal chamber of theenclosure; and collecting or depositing a material within the internalchamber of the incubator using the import/export tip. In variousembodiments of the method, the material may include a biologicalmicro-object. In some embodiments of the method, collecting ordepositing the material may include collecting or depositing thematerial within a well of a cell culture plate positioned within theinternal chamber of the incubator.

In various embodiments of the method, the method may further include thesteps of: withdrawing the import/export tip through one or more of thepassages between the exterior of the incubator and the internal chamberof the enclosure after collecting or depositing the material; and movingthe sealing element to a closed position such that the sealing elementcovers the plurality of openings in the enclosure.

In some embodiments of the method, the sealing element may be in theopen position for an amount of time which is sufficiently short so as toprevent the carbon dioxide content and/or the humidity of the airpresent in the internal chamber of the enclosure from equilibrating withthe carbon dioxide content and/or the humidity of the air surroundingthe incubator.

In various embodiments of the method, the method may further include thestep of actuating a sealing element actuator to move the sealing elementto the open position or closed position. In some embodiments, moving thesealing element between the open position and the closed position mayinclude sliding the sealing element relative to the enclosure. Invarious embodiments of the method, when the plurality of openings in thesealing element are in the open position, the plurality of openings inthe sealing element may be configured to be in register with a pluralityof wells in the cell culture plate.

In various embodiments of the method, the incubator may include asupport within the internal chamber of the incubator configured tosupport the cell culture plate. In various embodiments of the method,the method may further include the step of sliding the support, and acell culture plate resting on the support, from the internal chamber ofthe enclosure to a position outside of the internal chamber of theenclosure, thereby withdrawing the cell culture plate from the internalchamber of the enclosure. In some embodiments, sliding the support mayinclude sliding an access assembly comprising the support for the cellculture plate and an access door attached to the support. In someembodiments, sliding the support (or access assembly) may includesliding the support (or access assembly) along one or more tracks of anenclosure support that supports the enclosure. In some embodiments,sliding the support (or access assembly) may be performed by a humanoperator. In other embodiments, sliding the support (or access assembly)is performed robotically.

In various embodiments of the method, the method may further include thestep of sliding the support from the internal chamber of the incubatorto a position outside of the internal chamber of the enclosure, therebywithdrawing the support from the enclosure. In various embodiments ofthe method, the method may further include the step of placing a cellculture plate on the support while the support is in the positionoutside of the internal chamber of enclosure. In some embodiments,placing the cell culture plate may be performed by a human operator. Inother embodiments, placing the cell culture plate may be performedrobotically. In various embodiments of the method, the method mayfurther include the step of sliding the support, and the cell cultureplate placed upon the support, to a position inside the internal chamberof the enclosure. In some embodiments, sliding the support may includesliding an access assembly, wherein the access assembly comprises thesupport for the cell culture plate and an access door attached to thesupport. In various embodiments of the method, the step of sliding thesupport comprises sliding the support or access assembly along one ormore tracks of an enclosure support of the incubator. In someembodiments, sliding the support (or access assembly) may be performedby a human operator. In other embodiments, sliding the support (oraccess assembly) may be performed robotically.

In various embodiments, the method may further include the step ofestablishing an environment within the internal chamber of the enclosuresuitable for supporting a biological micro-object cultured in a cellculture plate positioned within the internal chamber of the enclosure.In various embodiments, the method may further include the step ofmeasuring one or more of a temperature, a humidity, and a carbon dioxidecontent of the internal chamber of the incubator. In variousembodiments, the method may further include the step of controlling oneor more of a temperature, a humidity, and a carbon dioxide content ofthe internal chamber of the incubator. In some embodiments, controllingthe temperature may include heating or cooling the internal chamber ofthe incubator. In some embodiments, controlling the humidity may includeproviding a humidity source to the internal chamber of the incubator. Insome embodiments, controlling the carbon dioxide content may includeproviding a gas source including carbon dioxide (e.g., a knownpercentage of carbon dioxide) to the internal chamber of the incubator.In some embodiments, the gas source comprising carbon dioxide mayfurther include oxygen and nitrogen. In some embodiments, providing agas source including carbon dioxide may include providing a purge gas tothe internal chamber.

In various embodiments, collecting or depositing the material isperformed with the import/export tip. In some embodiments, theimport/export tip comprises a plurality of tips, allowing substantiallysimultaneous collection of material from a plurality of wells of a cellculture plate or substantially simultaneous deposition of material intoa plurality of wells of the cell culture plate. Thus, in variousembodiments, the method may further include the step simultaneouslycollecting or depositing material from/into a plurality of wells in acell culture plate. In some embodiments, the collecting or depositingmay be performed robotically.

In some embodiments, the sealing element, when in the closed position,is capable of maintaining a pressure within the internal chamber that isgreater than the ambient air pressure. For example, the pressure withinthe internal chamber of the enclosure can be between about 0.0005 psi toabout 0.0100 psi above ambient pressure. Thus, in various embodiments,the method may further include the step of maintaining a pressure withinthe internal chamber of the enclosure at a pressure greater than apressure outside of the incubator when the sealing element is in aclosed position. In other embodiments, the method may further includethe step of maintaining a pressure within the internal chamber of theenclosure at a pressure greater than a pressure outside of the incubatorwhen the sealing element is in an open position. In some embodiments,maintaining a pressure within the internal chamber when the sealingelement is in an open position can include providing a purge gas to theinternal chamber.

In some embodiments, each of the plurality of openings in the enclosuremay have a diameter of about 1 mm to about 10 mm. In other embodiments,each of the plurality of openings in the enclosure has a diameter ofabout 1 mm to about 5 mm, or about 1.0 mm, about 1.5 mm, about 2.0 mm,about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm,about 5.0 mm, or any range defined by the foregoing values. In someembodiments, each of the plurality of openings in the sealing elementmay have a diameter of about 1 mm to about 10 mm. In some embodiments,each of the plurality of openings in the sealing element may have adiameter of about 1 mm to about 5 mm, or about 1.0 mm, about 1.5 mm,about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm,about 4.5 mm, about 5.0 mm, or any range defined by the foregoingvalues.

In yet another aspect of the invention, a method is provided foraccessing an internal chamber of an incubator, where the incubatorcomprises an enclosure having a plurality of openings and a sealingelement having more than one plurality of openings, wherein eachplurality of openings in the sealing element corresponds to at least asubset of the plurality of openings in the enclosure.

In various embodiments, the method includes the steps of: moving thesealing element to a first open position, thereby bringing a firstplurality of openings in the sealing element into register with a firstsubset of the plurality of openings in the enclosure, where the firstplurality of openings in the sealing element and the first subset of theplurality of openings in the enclosure provide a first plurality ofpassages from an exterior of the incubator to the internal chamber ofthe enclosure; advancing an import/export tip through one or more of thefirst plurality of passages between the exterior of the incubator andthe internal chamber of the enclosure; and collecting or depositing amaterial the internal chamber of the incubator using the import/exporttip. When the sealing element is in the first open position, anyopenings of the plurality of openings in the enclosure that are not inthe first subset of openings can be occluded by the sealing element. Invarious embodiments, the first plurality of passages may be configuredto be in register with a first subset of wells in a cell culture platepositioned within the internal chamber of the enclosure.

In various embodiments of the method, the method may further include thestep of moving the sealing element to a second open position, therebybringing a second plurality of openings in the sealing element intoregister with a second subset of the plurality of openings in theenclosure, the second plurality of openings in the sealing element andthe second subset of the plurality of openings in the enclosureproviding a second plurality of passages from an exterior of theincubator to the internal chamber. In some embodiments, the firstplurality of openings in the sealing element can be identical to thesecond plurality of openings in the sealing element. In otherembodiments, the first plurality of openings in the sealing element canbe different from the second plurality of openings in the sealingelement (e.g., the first and second pluralities of openings in thesealing element can be completely non-overlapping or partiallyover-lapping). When the sealing element is in the second open position,any openings of the plurality of openings in the enclosure that are notin the second subset of openings can be occluded by the sealing element.In various embodiments, the second plurality of passages may beconfigured to be in register with a second subset of wells in a cellculture plate positioned within the internal chamber of the enclosure.

In various embodiments of the method, the method may further include thestep of moving the sealing element to a third open position, therebybringing a third plurality of openings in the sealing element intoregister with a third subset of the plurality of openings in theenclosure, the third plurality of openings in the sealing element andthe third subset of the plurality of openings in the enclosure providinga third plurality of passages from an exterior of the incubator to theinternal chamber of the enclosure. In some embodiments, the thirdplurality of openings in the sealing element can be identical to thefirst and/or second pluralities of openings in the sealing element. Inother embodiments, the third plurality of openings in the sealingelement can be different from the first and/or second plurality ofopenings in the sealing element (e.g., the first, second, and thirdpluralities of openings in the sealing element can be completelynon-overlapping or partially over-lapping). When the sealing element isin the third open position, any openings of the plurality of openings inthe enclosure that are not in the third subset of openings can beoccluded by the sealing element. In various embodiments, the thirdplurality of passages may be configured to be in register with a thirdsubset of wells in a cell culture plate positioned within the internalchamber of the enclosure.

In some embodiments, the number of passages in the first plurality ofpassages may be the same as the number of wells in the cell cultureplate. In some embodiments, the number of passages in each of the first,second, and/or third plurality of passages may be equal to or less thanone-half, one-third, one-fourth, one-sixth, or one-twelfth the number ofwells in the cell culture plate.

In various embodiments of the method, the method may further include thestep of moving the sealing element to a closed position, therebybringing each of the plurality of the openings in the enclosure to anoccluded position.

In another aspect of the invention, an incubation system is provided.The incubation system can include: a well plate incubator, such asdescribed above or elsewhere herein; a robotic sampling componentconfigured to access the well plate incubator to remove/deliver samples;and at least one controller configured to open a plurality of passagesin the incubator and to control the robotic sampling component toaccess, via the plurality of passages, a plurality of wells of a wellplate contained within the well plate incubator. In various embodiments,the wells of the well plate may contain a biological material, includinga biological micro-object (e.g., a cell).

In some embodiments, the at least one controller may be furtherconfigured to close the plurality of passages. In some embodiments, thesystem may be configured to maintain the well plate incubator underpositive pressure. In some embodiments, the at least one controller maybe configured to control the robotic sampling component to withdraw amaterial from one of the plurality of wells of the well plate. In someembodiments, the at least one controller may be configured to controlthe robotic sample component to deliver the withdrawn material to amicrofluidic device. In some embodiments, the at least one controllermay be configured to control the robotic sample component to deliver thewithdrawn material to an analytical instrument. In some embodiments, theat least one controller may be configured to control the robotic samplecomponent to deliver one or more materials to one or more wells of thewell plate contained within the well plate incubator. In someembodiments, the one or more materials may be obtained from amicrofluidic device. In other embodiments, the one or more materials maybe obtained from an analytical instrument.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe claims that follow. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1B illustrate an isometric view and an exploded isometric viewof an incubator, respectively, in accordance with some embodiments.

FIGS. 2A-2C illustrate a top view of a lid, a printed circuit board andits associated connector, and an optional spacer, of an incubator,respectively, in accordance with some embodiments.

FIGS. 3A-3C illustrate an exploded isometric view showing the topsurface of a lid, a printed circuit board and its associated connector,and a spacer, respectively, that can be used in the embodiments ofincubators described herein.

FIGS. 3D-3F illustrate an exploded isometric view showing the topsurface of a lid, a sealing element, and a printed circuit board and itsassociated connector, respectively, that can be used in the embodimentsof incubators described herein.

FIGS. 4A-4B illustrate an exploded isometric view of bottom surfaces ofa lid and a printed circuit board and its associated connector,respectively, that can be used in some of the embodiments of incubatorsdescribed herein.

FIGS. 5A-B illustrate a lid having flexible tabs that can be part of theenclosures described herein. FIG. 5A illustrates a top surface of thelid, while FIG. 5B shows a view of the bottom surface of the lid.

FIGS. 5C-5E illustrate a top view of an incubator having the sealingelement in a closed position, a first open position, and a second openposition, respectively, in accordance with some embodiments.

FIGS. 5F-5G illustrate a top view of a portion of an incubator havingthe lid removed, with the sealing element in a closed position and anopen position, respectively, in accordance with some embodiments.

FIG. 5H illustrates a top view of a portion of an incubator having thelid and the sealing element removed and including a printed circuitboard, in accordance with some embodiments.

FIGS. 6A-6B illustrate a top view of a portion of an incubator havingthe sealing element in an open position and closed position,respectively, in accordance with some embodiments.

FIG. 7 illustrates an exploded isometric view of a portion of anincubator in accordance with some embodiments.

FIG. 8 illustrates an exploded isometric view of a portion of anincubator in accordance with some embodiments.

FIG. 9 illustrates a top view of a portion of an incubator in accordancewith some embodiments.

FIGS. 10A-10B illustrate a top view of a portion of an incubator, with asupport for a cell culture plate in an open and a closed position,respectively, in accordance with some embodiments.

FIG. 10C illustrates a top view of an enclosure support, in accordancewith some embodiments.

FIGS. 11A-11B illustrate views of a portion of a support for a cellculture plate of an incubator, in accordance with some embodiments.

FIG. 11C illustrates a partial side view of an incubator, in accordancewith some embodiments.

FIGS. 12A-12B illustrate a view of a magnet and a view of slide railsthat can be used in embodiments of the incubators disclosed herein.

FIG. 13 illustrates an embodiment of rails on a access assembly of anincubator, in accordance with some embodiments.

FIG. 14 illustrates an exploded view of an enclosure support of anincubator, in accordance with some embodiments.

FIG. 15 illustrates an exterior portion of an incubator, in accordancewith some embodiments.

FIG. 16 illustrates a side view of an incubator, in accordance with someembodiments.

FIGS. 17A-17B illustrate an isometric view of an incubator with asupport for a cell culture plate in an open and a closed position,respectively, in accordance with some embodiments.

FIG. 18 illustrates a schematic representation of a system forincubation having continuous access for export/import.

FIG. 19 is a graph of cell viability data obtained from cells culturedin an incubator of the invention during the first 24 hours after seeding(solid lines) and cells cultured entirely in a conventional incubator(dotted lines).

DETAILED DESCRIPTION

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Although the terms “first” and “second” may be used herein to describevarious features/elements, these features/elements should not be limitedby these terms, unless the context indicates otherwise. These terms maybe used to distinguish one feature/element from another feature/element.Thus, a first feature/element discussed below could be termed a secondfeature/element, and similarly, a second feature/element discussed belowcould be termed a first feature/element without departing from theteachings of the present invention.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical rangerecited herein is intended to include all sub-ranges subsumed therein.

As used herein, the term “micro-object” can encompass one or more of thefollowing: inanimate micro-objects such as microparticles; microbeads(e.g., polystyrene beads, Luminex™ beads, or the like); magnetic beads;microrods; microwires; quantum dots, and the like; biologicalmicro-objects such as cells (e.g., embryos, oocytes, sperm cells, cellsdissociated from a tissue, eukaryotic cells, protist cells, animalcells, mammalian cells, human cells, immunological cells, hybridomas,cultured cells, cells from a cell line, cancer cells, infected cells,transfected and/or transformed cells, reporter cells, prokaryotic cell,and the like); biological organelles; vesicles, or complexes; syntheticvesicles; liposomes (e.g., synthetic or derived from membranepreparations); lipid nanorafts (as described in Ritchie et al. (2009)“Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs,”Methods Enzymol., 464:211-231), and the like; or a combination ofinanimate micro-objects and biological micro-objects (e.g., microbeadsattached to cells, liposome-coated micro-beads, liposome-coated magneticbeads, or the like). Beads may further have other moieties/moleculescovalently or non-covalently attached, such as fluorescent labels,proteins, small molecule signaling moieties, antigens, orchemical/biological species capable of use in an assay.

As used herein, the term “cell” refers to a biological cell, which canbe a plant cell, an animal cell (e.g., a mammalian cell), a bacterialcell, a fungal cell, or the like. A mammalian cell can be, for example,from a human, a mouse, a rat, a horse, a goat, a sheep, a cow, aprimate, or the like.

As used herein, the term “maintaining (a) cell(s)” refers to providingan environment comprising both fluidic and gaseous components thatprovide the conditions necessary to keep the cells viable and/orexpanding.

As used herein, the term “expanding” when referring to cells, refers toincreasing in cell number.

As used herein, “import/export tip” refers to a mechanical deliverydevice sized to fit within one or more wells of a cell culture plate anddeposit/withdraw material and/or media. The import/export tip cancomprise, for example, a needle, a pin, or a similar structure having asurface capable of adhering to material and/or media located within orintended for the cell culture plate. The import/export tip can furthercomprise, for example, a hollow delivery tube having an internaldiameter sufficiently large to permit passage of material and/or medialocated within or intended for the cell culture plate. In someembodiments, the import/export tip may be made from a metal or ceramicmaterial. In some embodiments, the import/export tip may be made from apolymer (e.g., plastic). For example, the import/export tip can compriseplastic tubing, which may or may not be stiffened with an exteriorsleeve. In other embodiments, the import/export tip may be a cannula orneedle. An import/export tip may be any type of material that iscompatible with the material being transferred. The import/export tipmay be suitable for autoclaving or it may be disposable.

As used herein, a “microfluidic device” is a device that includes one ormore discrete microfluidic circuits configured to hold a fluid, eachcircuit comprised of interconnected circuit elements, including but notlimited to region(s), chamber(s), channel(s), and/or pen(s), and atleast two ports configured to allow the fluid (and, optionally,micro-objects suspended in the fluid) to flow into and/or out of themicrofluidic device. Typically, a microfluidic circuit of a microfluidicdevice will hold a volume of fluid of less than about 1 mL, e.g., lessthan about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8,7, 6, or 5 μL (or about 2-5, 2-10, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50,10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300μL).

As used herein, a “nanofluidic device” is a type of microfluidic devicehaving a microfluidic circuit that contains at least one circuit elementconfigured to hold a volume of fluid of less than about 1 μL, e.g., lessthan about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8,7, 6, 5, 4, 3, 2, 1 nL or less (or about 100 pL to 1 nL, 100 pL to 2 nL,100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pLto 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or1 to 50 nL).

As used here, reference numbers in the detailed description of theinvention refer not only to a specific embodiment, but are used forclarity and ease of review for the entire scope of the inventive matter.Specific embodiments of each element are shown in the figures and usethe same reference number, but such use is in no ways intended to limitthe breadth of the inventive matter to single embodiments.

Incubators and methods of using incubators are disclosed herein thatimprove accessibility to a cell culture plate in an internal chamberwithin an enclosure of the incubator, while also minimizing the chanceof contamination of the internal chamber of the incubator. Theincubators described herein can be more easily accessed by a robotic armor other tool, such as an import/export tip or other sampling devicethan conventional incubators that require opening a swinging lid or doorin order to access the internal chamber of the incubator. The lack of aswinging lid or door that exposes the internal chamber of the incubatorto the external environment can greatly decrease the chance ofcontamination of the incubator.

An incubator can include an enclosure having an internal chamberconfigured to support a cell culture plate having a plurality of wells.The enclosure can include a plurality of openings configured to allowaccess to the wells. The incubator can include a sealing elementconfigured to seal the plurality of openings in the enclosure. Thesealing element can include a first plurality of openings correspondingto at least a subset of the plurality of openings in the enclosure

Enclosure.

An incubator 100 includes an enclosure 102. The enclosure 102 caninclude a base 104 and a lid 106, 206 (see one exemplar in FIGS. 1A-1Band other exemplars in FIGS. 16 and 5C-5E). The base 104 and the lid106, 206 can define the internal chamber 110 of the incubator 100. Insome embodiments, the base 104, lid 106, 206, and a front plate 156 candefine the internal chamber 110 of the incubator 100. In someembodiments the base 104 can be formed from a rigid material having ahigh thermal conductivity and low thermal capacitance. Some suitablematerials can include aluminum, brass, ceramics or othercopper-containing alloys. Copper-containing alloys can be particularlyuseful due to antimicrobial properties conferred by the copper content.

The incubator 100 can further include an insulation material coupled tothe enclosure (See, for example, insulating panels 170 in FIGS. 7-8).The insulation material can be attached to one or more outer surfaces ofthe enclosure. A variety of plastics may be used to form insulatingpanels which may be coupled detachably or permanently to the exteriorwalls of the base 104 or may be fabricated for use as the lid. Forexample, one class of suitable insulating plastic may be amorphousthermoplastic polyetherimide, which is available in a wide range offormulations, and is available commercially as ULTEM™ (SABIC). Theinsulating panels may be formed to incorporate one or more recesses,where the recess includes air further insulating the enclosure. In someembodiments, the insulating panel may be about 1 mm, 2 mm, 3 mm, 4 mm, 5mm, 6 mm, 7 mm, 8 mmm, 9 mm, or about 10 mm thick. The insulating panelsmay be fabricated to create a recess between an outer surface of thepanel and the outer surface of the enclosure to which it is attached.For example, the insulating panels attached to the base 104 of theenclosure 102, may be fabricated to hollow out its inner surface,disposing the inner surface of the insulation panel about 1 mm, 2 mm, 3mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or about 11 mm away fromthe outer surface of the base 102, except where the panel 170 isattached to the base 104. This may create a pocket of air that is about1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or about 11mm thick at the sides of the base 104. The insulating panels may besuitable for autoclaving or may be removed from the enclosure prior toautoclaving.

Lid.

The lid 106, 206 can include an outer surface exterior to the enclosure102 and an inner surface within the enclosure 102 (See one exemplar inFIGS. 1A-1B and another exemplar in FIG. 16). The lid 106, 206 may bepart of a lid assembly 108 (See one exemplar in FIGS. 3A-C). The innersurface of the lid 106, 206 can include one or more recesses 124. Therecesses 124 in the lid 106, 206 can be configured to accommodate partsof the lid assembly 108, such as a printed circuit board (PCB) 132, 232and/or a spacer 134, each of which is described in additional detailbelow. In some cases, the recesses 124 can be configured to channel gasflow and/or provide insulation. In some embodiments the inner surfacecan include one or more recesses that can substantially surround groups213 of openings 212 (See one exemplar in FIG. 5B). Each group 213 caninclude two or more (e.g., 3, 4, 6, etc.) openings 212 of the pluralityof openings 212. The groups 213 of the openings 212 can improve a sealformed between the lid 206 and the sealing element 116, 216 when thesealing element 116, 216 is in the closed position. For example, theopenings 118, 218 in the sealing element 116, 216 can be occluded by thespace between the openings 212 of the groups 213. The openings 212 tothe left side of the groups 213 can form a first subset of openingswhile the openings 212 to the right side of the groups 213 can form asecond subset of openings. In some embodiments the recesses of the lid106, 206 can be sealed with a sealing material and/or insulatingmaterial. The sealing material can be configured to prevent air withinthe enclosure from filling the one or more recesses. Thus, the lid 106,206 can include a plurality of pockets filled with a gas orsubstantially lacking a gas (e.g., the pockets could include a vacuum orsub-atmospheric pressure). In some embodiments, the sealing material caninclude an adhesive layer adhered to the inner surface of the lid 106,206. The adhesive layer can include insulating materials. In someembodiments, the lid 106, 206 is made from a rigid insulating materialsuch as a polymer or plastic. In other embodiments, the lid 106, 206 ismade from a rigid material having a high thermal conductivity and lowthermal capacitance (e.g., aluminum, copper, brass, othercopper-containing alloys, or ceramics). One suitable class of plasticthat the lid may be made from is polyetherimide (e.g., ULTEM™), asdescribed above. The lid may be made from a material that can beautoclaved after use.

The lid 106, 206 can include one or more connectors configured tosealably connect the lid 106 to the base 104. Examples of the one ormore connectors include a magnet, a flexible tab, a flexible clip, orsimilar structures. In one example the lid includes flexible tabs 215that can be configured to engage with a pin 215 b to secure the lid 206to the base (See one exemplar in FIGS. 5C-5E). The seal between the base104 and the lid 106 does not have to be air-tight.

In some embodiments the lid can include an outer surface 207 (See oneexemplar in FIG. 5A) that includes a marking 207 a of an instructionsuch as “pull up to remove”. The lid 206 can also include instructions215 c on the compression tabs 215, such as “push to install”. Themarking 207 a and instructions 215 c can colored, etched, or adapted tobe machine readable by a computer imaging program.

The lid 106 and associated lid assembly 108 can include the plurality ofopenings 112 in the enclosure 102 providing access to the wells 120 ofthe cell culture plate 114.

Lid Assembly.

The lid assembly 108 of the incubator 100 can include a printed circuitboard (PCB) 132, 232 (See various exemplars in FIGS. 3B, 3F, 4B and 5H).The PCB 132 can be part of or coupled to the lid 106 of the incubator100. In another example, the PCB 132 can be positioned between theenclosure and the sealing element 216. For example, the PCB 132 can belocated between the sealing element 116 and an internal surface of a topof the enclosure 102, such as the lid 106. Alternatively, the PCB 232can be positioned proximal to (e.g., adjacent to) a top of the enclosure102 (e.g., the lid 206), with the sealing element interspersed betweenthe PCB 232 and the top of the enclosure 102 (See an exemplar in FIGS.3E-F). The PCB 132, 232 can have a substantially flat surface thatdirectly contacts a substantially flat surface of the sealing element116, 216 and/or a spacer 134. The PCB 132, 232 can include a pluralityof openings 138, 238 in register with the plurality of openings 112, 212of the lid 106, 206 to provide the openings of the enclosure 102. Insome embodiments the PCB 132, 232 includes one or more sensors on thePCB 132, 232. The one or more sensors can be selected from the groupconsisting of: a temperature sensor, a humidity sensor, an oxygensensor, and a carbon dioxide sensor. In yet other embodiments, the PCB132, 232 may include resistive heating elements, as described in moredetail below. The resistive heating elements can be located on a side ofthe PCB 132, 232 facing the internal chamber 110 of the enclosure 102and/or the cell culture plate 114. Alternatively, the resistive heatingelements can be located internally in the PCB 132, 232. The PCB 132, 232can include a multi-layer construction. The multi-layer construction caninclude the resistive heating elements internally such that theresistive heating elements are not exposed to the incubator environmentexternal to the PCB 132, 232. The multi-layer construction of the PCB132, 232 can improve the stiffness of the PCB 132, 232 and subsequentlyimprove the seal between the sealing element 116, 2116 and the PCB 132,232. When one or more sensors and/or resistive heating elements areincluded in the PCB 132, 232, each of these elements are located suchthat each element does not interfere with the openings 138, 238 in thePCB 132, 232. In some embodiments, the lid assembly 108 can be removablefrom the incubator. In some embodiments, the PCB may be designed to bedisposable after each use. The PCB 132, 232 may be connected to acontroller 174 (see one exemplar in FIG. 17) and/or other components viaa connector 136.

In some embodiments, the incubator 100 may include a spacer 134 as partof the lid assembly 108 (some exemplary embodiments are shown in FIGS.2C and 3C). The spacer 134 can be configured to reduce friction betweenthe sealing element 116, 216 and the PCB 132, 232 when the sealingelement 116, 216 moves between an open and a closed position. The spacer134 can have a plurality of openings 142. The plurality of openings 142on the spacer 134 can be in register with the plurality of openings 112,212 of the lid 106, 206 providing the openings of the enclosure 102. Theplurality of openings 142 on the spacer 134 can be in register with theplurality of openings 138, 238 on the PCB 132, 232. In variousembodiments, the plurality of openings 142 on the spacer 134 are inregister with the plurality of openings 112, 212 of the lid 106, 206 andin register with the plurality of openings 138, 238 on the PCB 132, 232.The spacer 134 can be located between the PCB 132, 232 and the sealingelement 116, 216. The spacer 134 may be configured to engage with thesealing element 116, 216, as the sealing element 116, 216 is movedbetween a closed position to any possible open positions. The spacer 134may be made of a compressible material such as rubber, silicone, orother polymeric materials which can reduce friction between the sealingelement 116, 216 and the PCB 132, 232. In some embodiments the spacer134 may be detachably assembled so that it may be autoclaved betweenuses. In other embodiments, the spacer 134 may be disposable after eachuse.

In some embodiments the spacer 134 is omitted from the lid assembly 108.In some embodiments, an outer surface of the PCB 132, 232 can be coatedwith Parylene™ by vapor deposition, which can protect the PCB fromabrasions caused by movement of the sealing element. Other types ofcoatings can be used on the PCB, such as urethane-based coatings andother chemicals, materials, and polymers that reduce friction betweenthe sealing element 116, 216 and the PCB 132, 232.

Openings in the Enclosure.

The number of openings in the enclosure 102, provided by the openings112, 212 in the lid 106, 206 (some exemplary embodiments are shown inFIGS. 1A-1B and FIGS. 5A-E) and openings (138, and optionally 142) ofthe associated lid assembly 108 (which may include the PCB 132, 232 and,optionally, spacer 134) may be the same as the number of the wells 120in the cell culture plate 114. The openings (112, 212, 138, 238, 142) ofthe lid 106, 206, PCB 132, 232, and optional spacer 134 may be inregister with the wells 120 in the cell culture plate 114 within theenclosure 102. In some other embodiments, the number of openings in theenclosure 102 may be different from the number of wells 120 in the cellculture plate 114. This may be used when more than one type of cellculture plate 114 is used in the incubator 100 and changing theenclosure 102 element to have fewer openings is not desired by theoperator.

In some embodiments, the enclosure 102 may have 96 openings. In otherembodiments, the enclosure 102 may have 384 openings. In someembodiments, the number of openings may be less than 96 or may be moreor less than 384. In some embodiments, the enclosure 102 may have 24 orfewer (e.g., 12 or 6) opening. In other embodiments, the enclosure 102may have 6 or fewer openings.

Base.

The base 104 can be configured with a hollow region forming part or allof the internal chamber 110 of the enclosure 102 (one exemplar shown inFIG. 1B and other exemplars are shown in FIGS. 10A and 17A). The base104 can include a bottom and four walls. The four walls can define thehollow region forming part or all of the internal chamber 110 of theenclosure 102. In some embodiments one of the four walls can have aheight that is shorter than the height of the other three walls. In someembodiments, the height of three of the four walls are the same. Thebase 104 may be made of a rigid material having a high thermalconductivity and low thermal capacitance, and be any of the suitablematerials described above. In one embodiment, the base 104 is made frombrass or another copper-containing alloy. The base 104 may haveinsulation panels attached, as described above, and may be autoclavedeither while assembled or upon partial or complete disassembly.

In some embodiments the base 104 and the lid 106 are formed of the samematerial. In other embodiments, the base 104 and lid 106 are formed fromdifferent materials.

Sealing Element.

The incubators 100 described herein include a sealing element 116 (oneexemplar is shown in FIG. 1B) and sealing element 216 (one exemplar isshown in FIGS. 5F-5G). The sealing element 116, 216 can be locatedinside the internal chamber 110 of the enclosure 102. For example, thesealing element 116, 216 can be configured to be located between thecell culture plate 114 and the lid 106, 206 of the incubator 100. Thesealing element 116, 216 can be configured to block the openings 112,212 in the lid 106, 206 of the incubator 100 which provide access towells 120 of a cell culture plate 114 inside the enclosure 102. Forexample, the sealing element 116, 216 can be configured to block,occlude, or obstruct a plurality of pathways between wells 120 in thecell culture plate 114 and the openings 112, 212 in the lid 106, 206.The sealing element 116, 216 can include one or more pluralities ofopenings 118, 218 which may each correspond to a portion or all of theplurality of openings in the enclosure 102. Access to the internalchamber 110 of the enclosure 102 and a cell culture plate 114 within, ifpresent, may be provided by positioning the sealing element 116, 216such that one or more of the plurality of openings (112, 212, 138, 238,and optionally 142) in the enclosure 102 is in register with one or moreopenings 118, 218 in the sealing element 116, 216.

The sealing element 116, 216 can be made of a wide variety of materials,including metals or plastic. Examples of suitable metals and plasticsinclude aluminum, brass, and polymers such as ULTEM, PEEK, Teflon, etc.The use of a metal sealing element can improve heat transfer and reducethe likelihood of condensation forming or collecting on the sealingelement. In some embodiments, the sealing element 116, 216 is made ofaluminum or brass, an inexpensive alternative which allows the sealingelement 116, 216 to be disposable or suitable for autoclaving. In otherembodiments, a plastic material may be used, which also permitsdisposability or tolerance to autoclaving.

The sealing element 116, 216 can be movable between a closed position inwhich the sealing element 116, 216 covers the plurality of openings(112, 212, 138, 238, and optionally 142) in the enclosure 102, and anopen position in which the plurality of openings 118, 218 of the sealingelement 116, 216 are in register with at least a portion of theplurality of openings (112, 212, 138, 238, and optionally 142) in theenclosure 102. The plurality of openings 118, 218 in the sealing element116, 216 can be configured to be in register with the plurality of wells120 in the cell culture plate 114 within the enclosure 102.

In some embodiments, a first plurality of openings 118, 218 in thesealing element 116, 216 can be the same number as the plurality of theopenings (112, 212, 138, 238, and optionally 142) in the enclosure 102.In other embodiments, a first plurality of openings 118, 218 in thesealing element 116, 216 can be a number less than the plurality of theopenings (112, 212, 138, 238, and optionally 142) in the enclosure 102.In some embodiments, the number of openings in the first plurality ofopenings in the sealing element 116, 216 is one-half, one-third,one-fourth, one-sixth, one-twelfth, or fewer the number of openings(112, 212, 138, 238, and optionally 142) in the enclosure 102.

FIGS. 5C-E illustrate first and second subsets of openings 212 a, 212 b(including 238 and optionally 142) in the enclosure 102 which canprovide access to a cell culture plate 114 located within the enclosure102 when the sealing element 116, 216 is in a first open position and asecond open position, respectively. FIG. 5C illustrates the sealingelement 216 in a closed position such that the openings 212 of the lid206 are occluded. FIG. 5D illustrates the sealing element 216 in a firstopen position such that a first plurality of openings 218 (not shown) inthe sealing element 216 are in register with the rows of openings 212 aof the lid 206, resulting in the rows of openings 212 a being open,while the rows of openings 212 b are occluded. FIG. 5E illustrates thesealing element 216 in a second open position such that the firstplurality of openings 218 (not shown) in the sealing element 218 are inregister with the rows of openings 212 b of the lid 206, resulting inthe rows of openings 212 b being open, while the rows of openings 212 aof the lid 206 are occluded.

In some embodiments, the sealing element 116, 216 may further have asecond plurality of openings 118, 218 which may be different from thefirst plurality of openings 118, 218. For example, the second pluralityof openings 118, 218 may be in a physically different location from thefirst plurality of openings 118, 218. The first and second plurality ofopenings 118, 218 may correspond to subsets of the plurality of openings(112, 212, 138, 238, and optionally 142) in the enclosure 102, e.g., thenumber of openings in the first and/or second plurality of openings 118,218 in the sealing element 116, 216 may be fewer than the number ofopenings (112, 212, 138, 238, and optionally 142) in the enclosure 102.In some embodiments, the number of openings in the first and/or secondplurality of openings 118, 218 in the sealing element 116, 216 isone-half, one-third, one-fourth, one-sixth, one-twelfth, or fewer thenumber of openings (112, 212, 138, 238, and optionally 142) in theenclosure 102.

In some embodiments, the sealing element 116, 216 may further have athird plurality of openings 118, 218, which may be different from thefirst and/or second plurality. For example, the third plurality ofopenings 118, 218 may be in a physically different location from thefirst plurality and/or second plurality of openings 118, 218. In someembodiments the number of openings in the third plurality of openings118, 218 in the sealing element 116, 216 is one-half, one-third,one-fourth, one-sixth, one-twelfth, or fewer the number of openings(112, 212, 138, 238, and optionally 142) in the enclosure 102. In someembodiments, the number of openings in the first, second, and/or thirdplurality of openings 118, 218 in the sealing element 116, 216 isone-half, one-third, one-fourth, one-sixth, one-twelfth, or fewer thenumber of openings (112, 212, 138, 238, and optionally 142) in theenclosure 102.

When the sealing element 116, 216 is moved between a closed position, inwhich the sealing element 116, 216 covers the plurality of openings(112, 212, 138, 238, and optionally 142) in the enclosure 102, to thefirst open position, then the first plurality of openings 118, 218 ofthe sealing element 116, 216 come into register with a first subset ofthe plurality of openings (112, 212, 138, 238, and optionally 142) inthe enclosure 102, and all other openings in the enclosure 102 that arenot in the first subset are occluded. When the sealing element 116, 216has a second plurality of openings 118, 218, the sealing element 116,216 may be further moved from the closed position or the first openposition to a second open position, wherein the second plurality ofopenings 118, 218 of the sealing element 116, 216 come into registerwith a second subset of the plurality of openings (112, 212, 138, 238,and optionally 142) in the enclosure 102, and all other openings in theenclosure 102 that are not in the second subset are occluded. When thesealing element 116, 216 has a third (or further) plurality of openings118, 218 the scaling element 116, 216 may be moved from the closedposition, first open position, or the second open position to a third(or further) open position, wherein the third (or further) plurality ofopenings 118, 218 in the sealing element 116, 216 come into registerwith a third (or further) subset of the plurality of openings (112, 212,138, 238, and optionally 142) in the enclosure 102, and all otheropenings in the enclosure 102 that are not in the third (or further)subset are occluded. The subset of the openings (112, 212, 138, 238, andoptionally 142) in the enclosure 102 opened by moving the sealingelement 116, 216 to the first, second, third, or further open positionmay be non-overlapping with the subset of openings (112, 212, 138, 238,and optionally 142) in the enclosure 102 opened by moving the sealingelement 116, 216 to one or all of the other open positions.

Size(s) of Openings in the Enclosure and the Sealing Element.

The plurality of openings (112, 212, 138, 238, and optionally 142) inthe enclosure 102 (which includes openings in the lid 106, 206 and theopenings in the elements making up the lid assembly 108) and the one ormore pluralities of openings 118, 218 in the sealing element 116, 216can be sized to allow for an import/export tip to access individualwells 120 in the cell culture plate 114. In some cases the openings canbe sized to correspond to the size and shape of the wells 120 in thecell culture plate 114. In other embodiments, the plurality(ies) ofopenings (112, 212, 138, 238, and optionally 142) in the enclosure 102and openings 118, 218 of the sealing element 116, 216 can be sized justsufficiently large to permit the import/export tip to access theindividual wells 120 in the cell culture plate 114 without necessarilybeing of the same size or shape as the wells 120. For instance anopening may be octagonal while the well 120 may be round or the openingmay be slightly smaller than the well 120, while still permitting theimport/export tip to access the well 120. In some embodiments theopenings (112, 212, 118, 218, 138, 238, and optionally 142) can be sizedto restrict the vapor phase in the internal chamber 110 of the incubator100 from passing through the openings to the exterior of the incubator100.

In various embodiments, the plurality(ies) of openings (112, 212, 118,218, 138, 238, and optionally 142) in the enclosure 102 and/or thesealing element 116 may independently have a diameter of about 1.0 mm,1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm,2.0 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3.0 mm, 3.2 mm, 3.4 mm, 3.6 mm,3.8 mm, 4.0 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5.0 mm, 5.2 mm, 5.4 mm,5.6 mm, 5.8 mm, 6.0 mm, 6.2 mm, 6.4 mm, 6.6 mm, 6.8 mm, 7.0 mm, 7.2 mm,7.4 mm, 7.6 mm, 7.8 mm, 8.0 mm, 8.2 mm, 8.4 mm, 8.6 mm, 8.8 mm, 9.0 mm,9.2 mm, 9.4 mm, 9.6 mm, 9.8 mm, or about 10.0 mm. In some embodimentsthe plurality(ies) of openings in the enclosure 102 and/or the sealingelement 116, 216 may independently have a diameter of about 1 mm toabout 10 mm. In some embodiments the plurality(ies) of openings in theenclosure 102 and/or the sealing element 116, 216 may independently havea diameter of about 1 mm to about 5 mm. In some embodiments theplurality(ies) of openings in the enclosure 102 and/or the sealingelement 116, 216 may independently have a diameter of about 1.5 mm toabout 4.5 mm. In some embodiments the plurality(ies) of openings in theenclosure 102 and/or the sealing element 116, 216 may independently havea diameter of about 1.7 mm to about 4.0 mm. In some embodiments theplurality(ies) of openings in the enclosure 102 and/or the sealingelement 116, 216 may independently have a diameter of about 1.7 mm toabout 1.8 mm. In some embodiments the plurality(ies) of openings in theenclosure 102 and/or the sealing element 116, 216 may independently havea diameter of less than about 10 mm and more than about 1 mm. In someembodiments the plurality(ies) of openings in the enclosure 102 and/orthe sealing element 116, 216 may independently have a diameter of lessthan about 5 mm and more than about 1 mm. In some embodiments theplurality(ies) of openings in the enclosure 102 and/or the sealingelement 116, 216 may independently have a diameter of less than about 4mm and more than about 1 mm. In some embodiments the plurality(ies) ofopenings in the enclosure 102 and/or the sealing element 116, 216 mayindependently have a diameter of less than about 3 mm and more thanabout 1 mm. In some embodiments the plurality(ies) of openings in theenclosure 102 and/or the sealing element 116, 216 may independently havea diameter of less than about 2 mm and more than about 1 mm.

The diameter of each of the plurality(ies) of openings (112, 212, 118,218, 138, 238, and optionally 142) in the enclosure 102 and/or thesealing element 116 can be selected based on the process conditions andthe properties of the incubator 100 and cell culture plate 114 used withthe incubator 100. Examples of process conditions and properties of theincubator 100 include: size and number of openings (112, 212, 118, 218,138, 238, and optionally 142), a desired vapor flow rate through theopenings, cell culture plate 114 configuration including number of wells120, a desired positive pressure operating range for the internalchamber 110 of the incubator 100, purge gas composition, etc. Forexample, when a cell culture plate 114 with 96 wells 120 is used withthe incubator 100, the openings (112, 212, 118, 218, 138, 238, andoptionally 142) in the enclosure 102 and/or the sealing element 116, 216can each be sized with a diameter of about 1.5 mm to about 4 mm, about1.7 mm to about 4 mm, or about 1.726 mm to about 4 mm. For example, whena cell culture plate 114 with 384 wells 120 is used with the incubator100, the openings (112, 212, 118, 218, 138, 238, and optionally 142) inthe enclosure 102 and/or the sealing element 116, 216 can each be sizedwith a diameter of about 1.5 mm to about 2.5 mm, about 1.7 mm to about2.0 mm, or about 1.726 mm to about 1.8 mm.

The plurality of openings (112, 212, 138, 238, and optionally 142) inthe enclosure 102 may have the same diameter size as the size of thediameter of the one or more pluralities of openings 118, 218 in thesealing element 116, 216. The plurality of openings (112, 212, 138, 238,and optionally 142) in the enclosure 102 and the one or more pluralitiesof openings 118, 218 in the sealing element 116, 216 can includemultiple different sizes of openings. A first subset of theplurality(ies) of openings of the enclosure 102 and/or the sealingelement 116, 216 can have a first size. A second subset of theplurality(ies) of openings of the enclosure 102 and/or the sealingelement 116, 216 can have a second size. In some cases, a third subsetof the plurality(ies) of openings of the enclosure 102 and/or thesealing element 116, 216 can have a third size. The first size, secondsize, and third size can be different. In some embodiments, the size ofthe openings (112, 212, 138. 238, and optionally 142) of the enclosure102 is a first size and the size of the openings 118, 218 in the sealingelement 116, 216 is a second size, where the second size is differentfrom the first size, as long as the import/export tip can enter.

The seal between the sealing element 116, 216 and other structures inthe incubator 100 does not have to be air tight. The seal can beconfigured to allow some gas flow from the internal chamber 110 throughthe openings (112, 212, 138, 238, and optionally 142) to the exterior ofthe incubator 100. For example, a pressurized gas source can be providedto provide a purge gas to the internal chamber 110. A small gas flow ofthe purge gas can pass through the internal chamber 110 of the enclosure102 and exit through the openings 112, 212 in the lid 106, 206 while thesealing element 116, 216 is in the closed position to seal the openings112, 212 in the lid 106, 206. In some embodiments a small positivepressure can be maintained within the internal chamber 110 to prevent orminimize the chance of contamination of the environment in the internalchamber 110. In some embodiments the sealing element 116, 216 isconfigured to form a seal with the plurality of openings (112, 212, 138,238, and optionally 142) in the enclosure 102 that allows the enclosure102 to maintain a pressure between about 0.0005 psi to about 0.01000 psiabove ambient pressure in the internal chamber 110 when gas from thepressurized gas source flows into the internal chamber 110.

Internal Chamber of the Enclosure.

The internal chamber 110 volume can be varied to accommodate a cellculture plate 114 having a desired size. In some embodiments theinternal chamber 110 has a volume of about 50 cm³ to about 300 cm³. Insome embodiments the internal chamber 110 has a volume of about 100 cm³to about 500 cm³. In some embodiments the internal chamber 110 has avolume of about 200 cm³ to about 750 cm³. In some embodiments theinternal chamber 110 has a volume of about 400 cm³ to about 1,000 cm³.In some embodiments the internal chamber 110 has a volume of about 500cm³ to about 1500 cm³. In some embodiments the internal chamber 110 hasa volume of about 750 cm³ to about 2000 cm³.

An incubator 100 as described herein can accommodate cell culturesplates of varying sizes within the enclosure 102. In some embodimentsthe cell culture plate 114 is a 96-well plate. The 96-well plate canhave an 8 well by 12 well configuration. In some embodiments the cellculture plate 114 is a 384-well plate. In some embodiments the cellculture plate 114 can have less than 96 wells. For example a cellculture plate 114 with 12 or fewer wells can be used. In someembodiments the cell culture plate 114 has 6 or fewer wells. The cellculture plates may have either a rounded bottom, which includes aU-shaped bottom, V-shaped, or flat shaped bottom to each of the wells inthe culture plate.

Sealing Element Actuator.

The incubator 100 can include a sealing element actuator 144 (anexemplar is shown in FIGS. 6A-B and another exemplar is shown in FIGS.14 and 16). The sealing element actuator 144 can be configured to movethe sealing element between an open and a closed position. The sealingelement actuator 144 can include a motor or rotary solenoid or similaractuator. For example, in some cases a stepper motor could be used. Thestepper motor can, for example, operate with a 0.5 hz frequency.Alternatively, a rotary solenoid can be used that operates with a 60 hzfrequency.

In some embodiments, the actuator 144 may be configured to move thesealing element 116/216 between the closed position and an openposition. In some embodiments the actuator 144 can be configured to movethe sealing element 116, 216 between the closed position and a pluralityof open positions. For example, the actuator 144 can be configured tomove the sealing element 116, 216 between the closed position and afirst open position and a second open position. In some embodiments theactuator 144 can be further configured to move the sealing element 116,216 to a third (or further) open position. The actuator 144 may beconfigured to move the sealing element 116, 216 between a first openposition and the closed position. The actuator 144 may then beconfigured to move the sealing element 116, 216 between a second openposition and the closed position. The actuator 144 may further beconfigured to move the sealing element 116, 216 between a third openposition and the closed position.

The incubator 100 can be configured to maintain a selected internaltemperature, humidity, and gas content within the internal chamber 110of the enclosure 102. The incubator 100 can include a controller 174 (anexemplar is shown in FIG. 18) configured to maintain the selectedinternal temperature, humidity, and gas content within the internalchamber 110 of the enclosure 102. The internal temperature, humidity,and gas content within the internal chamber 110 of the enclosure 102 canbe selected to maintain the materials within the incubator 100. In someembodiments, the temperature may be maintained in a range from about 4°C. to about 40° C. In some embodiments the temperature can be maintainedbetween about 4° C. to about 39° C., about 15° C. to about 39° C., about20° C. to about 38° C., about 25° C. to about 38° C., or about 30° C. toabout 38° C. In some embodiments the relative humidity is maintainedabove about 60%, 70%, 80%, or above about 90%. In some embodiments, therelative humidity is maintained at about 70%, 75%, 80%, 85%, 90%, orabout 95%. In some embodiments the carbon dioxide content is maintainedaround 1%, 2%, 3%, 4% or around 5%.

In some embodiments the incubator 100 may include a temperaturecontroller 174 (an exemplar is shown in FIG. 18) configured to maintaina temperature of the internal chamber 110 within a desired range (FIG.2B, 3B, 4B). The incubator 100 can include a first heating/coolingdevice engaged with a bottom of the enclosure 102, such as the base 104.Alternatively, the first heating/cooling device can be engaged with athermally conductive layer that contacts directly or indirectly with theenclosure 102 of the incubator 100. The first heating/cooling device canprovide heating or cooling to the thermally conductive layer. Thethermally conductive layer can provide heating or cooling to a portionof the enclosure 102. The thermally conductive layer can be made of athermally conductive material (e.g., aluminum, copper, brass, othercopper-containing alloys, or a ceramic), as discussed above. The use ofthe thermally conductive layer can improve the uniformity of the heattransfer to the enclosure 102. The first heating/cooling device can becontrolled by the temperature controller 174. Examples ofheating/cooling devices include: a resistive heater, a fluid coilconfigured to circulate a heat exchange fluid, one or more Peltierdevices, and the like. In some embodiments, the fluid coil includesaccess ports on its exterior to permit entry of fluid forcooling/heating by a Peltier device. In some embodiments having a fluidcoil, the heating/cooling device is a separate component from the base104. In embodiments having a heating/cooling device separable from thebase, disassembly is possible to permit autoclaving. In otherembodiments, the first heating/cooling device can be integral with theenclosure 102. For example, a fluid coil can be integral with the base104 of the enclosure 102. The temperature controller 174 can include orreceive input from one or more temperature sensors. The temperaturesensors can be attached to the PCB 132, 232, a portion of the enclosure102 (e.g., the base 104), and/or the first heating/cooling device.Examples of temperature sensors include thermistors and/or integratedcircuits. Integrated circuits can have less electrical noise and anaccuracy of +/−0.25° C. without the need for calibration.

The first heating/cooling device can directly contact (or indirectlyprovide heat transfer to) an outer surface of the bottom of theenclosure 102, such as the base 104. In some embodiments the firstheating/cooling device contacts at least about 75% of the outer surfaceof the bottom of the enclosure 102. In some embodiments the firstheating/cooling device contacts at least about 80% of the outer surfaceof the bottom of the enclosure 102. In some embodiments the firstheating/cooling device contacts at least about 85% of the outer surfaceof the bottom of the enclosure 102. In some embodiments the firstheating/cooling device contacts at least about 90% of the outer surfaceof the bottom of the enclosure 102. In some embodiments the firstheating/cooling device contacts at least about 95% of the outer surfaceof the bottom of the enclosure 102. In some embodiments, the firstheating/cooling device can maintain a temperature in the internalchamber 110 of the enclosure 102 in a range from about 4° C. to about40° C., about 4° C. to about 39° C., about 4° C. to about 38° C., orabout 4° C. to about 37° C. In some embodiments the temperature can bemaintained between about 10° C. to about 37° C., about 15° C. to about39° C., about 20° C. to about 38° C., about 25° C. to about 38° C.,about 30° C. to about 38° C. In other embodiments, the firstheating/cooling device can maintain a temperature in the internalchamber 110 of the enclosure 102 at about 4° C., 5° C., 6° C., 7° C., 8°C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17°C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26°C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35°C., 36° C., 37° C., 38° C., 39° C., or 40° C. In some embodiments theinternal chamber 110 of the enclosure is maintained at about 37° C.

The incubators 100 described herein can include a second heating/coolingdevice engaged with a top of the enclosure 102, such as a portion of thelid 106, 206 or lid assembly 108 or the PCB 132, 232. The secondheating/cooling device can be within the enclosure 102. Examples ofheating/cooling devices include: a resistive heater, a fluid coilconfigured to circulate a heat exchange fluid, and one or more Peltierdevices, and the like. In some embodiments, the second heating/coolingdevice may be a resistive heater. In some embodiments, the secondheating/cooling device is part of the PCB 132, 232, which is engagedwith the top of the enclosure 102, such as the lid 106, 206. The secondheating/cooling device can be controlled by the temperature controller174 (FIG. 18). The second heating/cooling device can include a pluralityof openings 138, 238 that are in register with the plurality of openings(112, 212, 138, 238, and optionally 142) in the enclosure 102. Thesecond heating/cooling device can include resistive heating elementsthat are part of the PCB 132, 232. The resistive heating elements 140can be located on a side of the PCB 132, 232 facing the internal chamber110 of the enclosure 102 and/or the cell culture plate 114 (FIG. 4B) orinternal to the PCB 132, 232. In some embodiments, the secondheating/cooling device can maintain a temperature in a range from about4° C. to about 40° C., about 4° C. to about 39° C., about 4° C. to about38° C., or about 4° C. to about 37° C. In some embodiments thetemperature can be maintained between about 10° C. to about 37° C.,about 15° C. to about 39° C., about 20° C. to about 38° C., about 25° C.to about 38° C., or about 30° C. to about 38° C. In various embodiments,the second heating/cooling device may maintain a temperature that is0.1° C., 0.2° C., 0.3° C., 0.4° C., 0.5° C., 0.6° C., 0.7° C., 0.8° C.,0.9° C., 1.0° C., 1.1° C., 1.2° C., 1.3° C., 1.4° C., 1.5° C., 1.7° C.,1.9° C., 2.0° C., 2.3° C., 2.5° C., 2.7° C., 2.9° C., 3.0° C., 3.3° C.,3.5° C., 3.7° C., 3.9° C., 4.0° C., 4.3° C., 4.5° C., 4.7° C., 4.9° C.or 5.0° C. higher than the first heating/cooling device. When the secondheating/cooling device maintains a temperature that is higher than thetemperature maintained by the first heating/cooling device, condensationnear the top of the internal chamber 110, and hence on the cell cultureplate 114, may be prevented.

Cell Culture Plate Support.

The incubators 100 disclosed herein can include a support 122, 222 forthe cell culture plate 114, 224 (an exemplar is shown in FIG. 1B, FIGS.7-8 and in FIGS. 10A-10B). The cell culture plate support 122, 222 canbe configured to slideably move relative to the enclosure 102 from aposition within the enclosure 102 to a position outside of the internalchamber 110 of the enclosure 102. The illustrated supports 122, 222 havea T-shape, although other shapes can be used to support the cell cultureplate 114. The support 122, 222 for the cell culture plate 114 can beattached to an access door 154 on the incubator 100, either directly orengaged through one or more intervening structures or parts, such as thebiased connections 255 (FIGS. 11A-11C). The culture plate support 122,222 may be made of a plastic. In some embodiments, the culture platesupport 122, 222 may be made of metal, which may be brass, in onenon-limiting example. The support can also include a distal lip 223 onthe support 222 for the cell culture plate 114 (See one exemplar inFIGS. 17A-17B).

In some embodiments the support 122, 222 for the cell culture plate 114and the access door 154, 254 can form an access assembly 168, 268 (anexemplar is shown in FIG. 8 and in FIGS. 11A-11B). The access assembly168, 268 can include a front plate 156, 256 that sealably interfaceswith a portion of the enclosure 102 as shown in FIGS. 7-9 and 10A. Theaccess assembly 168, 268 can include a floating connection between thefront plate and the access door. For example, a biased connection 255between the front plate and the access door can be configured to providea compressive force to the front plate to seal the front plate relativeto a portion of the enclosure 102 (See one exemplar in FIGS. 11A-11B).The access assembly may be configured such that the cell culture support122, 222 maintains the cell culture plate 114 at a height within theenclosure 102 such that the passages 150A introducing the environmentalor purging gas(es) are at the same height, relative to the bottom andtop of the enclosure, as the cell culture plate 114. (See descriptionbelow.) Keeping the gas entry at the same level as the cell cultureplate provides optimized humidity control, optimized gas circulation andprevents condensation on the cell culture plate. Accordingly the supportnotch 146 may be configured to support the side of the cell culturesupport at the same height at the passages for gas 150A. In someembodiments, the top of the cell culture plate 114 is at least about 2mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, or about 20 mm from thetop of the internal chamber 110 of the enclosure 102. In someembodiments, the lower surface of the cell culture plate support is atleast about 0 mm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm,or about 10 mm from the bottom inner surface of the internal chamber 110of the enclosure 102.

The access assembly 168, 268 may include track guides 166 or rails 266to permit the access assembly 168, 268 to slide open or shut, permittingaccess to the cell culture plate 114. The track guides 166 or rails 266can be configured to slide relative to tracks 162, 262 on an enclosuresupport 160, 260. The movement of the access assembly 168, 268 may bedirected by a controller 174 (See FIG. 18), which may be included aspart of incubator 100.

The access assembly 168, 268 can be movably mounted on an enclosuresupport 160, 260 that also supports the enclosure 102. The enclosuresupport 160 can include tracks 162 such that the access assembly 168 mayslide on track guides 166 relative to the tracks 162 on the enclosuresupport 160. The track guides 166 can have various cross-sectionalshapes. In one example the track guides can have a flat surface with arectangular or square cross sectional shape as shown in FIG. 8. In someembodiments the track guides 166 can have a circular or roundedcross-sectional shape such as the rails 266 illustrated in FIGS. 10B,11A, and 11B. The front plate 156, 256 of the access door 154, 254 maybe made of a metal or a plastic, similarly to the materials describedabove as suitable for the enclosure 102. In some embodiments, the frontplate 156, 256 is made of a material having high thermal conductivity(e.g., aluminum, copper, brass, a copper-containing alloy, or aceramic). The access door 154, 254 may be made of metal or plastic, andmay have a handle 172, 272. The culture plate support may be detachablefrom the access door 154, 254 for cleaning, for example, by autoclaving.The front plate 156, 256 and access door 154, 254 may also be cleaned byautoclaving, either as assembled or with disassembly.

The track guides/rails of the access assembly 168, 268 can include oneor more stops or engagement surfaces to help hold the access assembly168, 268 in one or more discrete positions, such as open and closedpositions. The track guides 166 or rails 266 can include an engagementsurface configured to engage with a complementary structure of theenclosure support 160, 260 to secure a position of the access assembly168, 268 relative to the enclosure support 160, 260. The position of theaccess assembly 168, 268 can correspond to an open or closed position ofthe access assembly 168, 268. In some embodiments the incubator caninclude a door switch configured to mechanically, electronically, ormagnetically engage with a complementary structure of the accessassembly 168, 268.

In some embodiments the support 122 for the cell culture plate 114 canbe one or more internal surfaces or features mounted on or fabricated aspart of the internal surface of the enclosure 102. For example, one ormore projections can extend from the sides of the enclosure 102 towardsthe internal chamber 110 to support the cell culture plate 114 withinthe internal chamber 110. In another example, the internal surface ofthe enclosure 102 may be notched to provide a resting support 146, 246,247 (exemplars shown in FIGS. 7, 17A, and 17B) for a portion of theculture plate support 122, 222 opposite to its attachment to the frontplate 156, 256 (an exemplar is shown in FIG. 7). When the cell cultureplate 114 is placed on the support 122, 222 within the enclosure 102,the openings (112, 212, 138, 238, and optionally 142) in the enclosure102 may be in register with the wells 120 in the cell culture plate 114.

The incubator 100 can include at least one passage 150A in the enclosure102 configured for gas entry (an exemplar is shown in FIGS. 7-8). Thepassage can be used to supply a purge gas or other gas selected tomaintain the desired internal environment within the incubator 100. Insome embodiments, the gas entry passage(s) 150A may be formed through awall of the base 104. In some embodiments, the gas entry passage(s) 150Ais at least about 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm,11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, or 20 mmfrom the top of the internal chamber 110 of the enclosure 102. In someembodiments, the gas entry passage(s) 150A are located at a height on aside of the base that is equivalent to the height of a side of the cellculture plate 114 when supported within the enclosure 102. The gas maybe provided to maintain a positive pressure within the internal chamber110. For example, the pressure of the internal chamber 110 can bemaintained between about 0.0005 psi to about 0.01000 psi above ambientpressure. Clean rooms typically use a positive pressure of about 0.0072psi or less. In some embodiments the pressure of the internal chamber110 can be maintained less than about 0.0072 psi above ambient pressure.In some embodiments the pressure of the internal chamber 110 can bemaintained above about 0.0072 psi above the ambient pressure. In somecases, the flow rate of the gas can be less than or about 10 liters perhour, 9 liters per hour, 8 liters per hour, 7 liters per hour, 6 litersper hour, 5 liters per hour, 4 liters per hour, 3 liters per hour, 2liters per hour, or 1 liter per hour. The flow rate may be more thanabout 0.5 liter per hour. In some embodiments the flow rate can be about1 liter/hour to about 10 liters/hour.

The purge or environmental gas may be conditioned to provide a desiredhumidity as well as a desired mixture of gases. In some embodiments theenvironmental gas is conditioned to provide a relative humidity aboveabout 50%, 60%, 70%, 75%, 80%, 85%, or above about 90%. In someembodiments, the gas is conditioned to provide a relative humidity ofabout 70%, 75%, 80%, 85%, 90%, or about 95%. In some embodiments theenvironmental gas is conditioned to provide a carbon dioxide content ofaround 1%, 2%, 3%, 4% or around 5%.

In some embodiments the internal chamber 110 of the enclosure 102 caninclude a reservoir configured to hold a fluid, such as a fluidreservoir. The incubator 100 can include at least one fluid drainpassage 150B in the enclosure 102 configured to drain the fluidreservoir within the enclosure 102 or drain the enclosure 102 itself (anexemplar is shown FIGS. 7-8). In some cases, the fluid can be used toprovide humidity to the internal chamber 110. In some cases, the fluidcan be used to control the temperature of the internal chamber 110. Insome embodiments, the fluid drain passage 150B may be formed through aside wall of the base 104. The fluid drain passage 150B can be sealable.

The incubator 100 may include electrical connections 152, 252 to powerthe sealing actuator 144, heat and cool the enclosure 102, open and shutthe culture plate support 122, 222, and/or operate the sensors of thePCB 132, 232, amongst other operations (an exemplar is shown in FIG. 8and another in FIG. 14). The electrical connections 152, 252 may beconnected to the first heating/cooling device contacting the bottom sideof the base 104, enclosure support 160, 260, enclosure 102, PCB 132,232, sealing element 116, 216 or other components of the lid assembly108.

The incubator 100 can include an enclosure support 160, 260 configuredto support the enclosure 102 (See one exemplar in FIGS. 7-8 and anotherexemplar in FIG. 14). One or more adjustable connectors on support legs164 can be configured to connect the enclosure support 160 to theenclosure 102.

The incubators described herein can be configured to reduce or minimizethe formation of condensation within the enclosure or on other parts ofthe incubator. The lid 106, 206 can be designed to minimize theformation of condensation on parts of the lid while also making the lideasy to clean using autoclaving or other cleaning methods. In oneexample, the lower side of the lid can have some of the recesses filledwith a foam cell polymer that is in register with the openings 112, 212of the lid 106, 206. In another example, portions of the recesses can befiled with materials such as a hydrophobic material like Parylene™and/or with Kapton™ sheets or tapes. In yet another example, some of therecesses could be sealed using a thin metal sheet. In yet anotherexample the structure of the lid could be inverted so that the texturedside faces the exterior of the incubator. In another example theinterior surface of the lid 106, 206 can be shaped to include additionalflat section to improve sealing. In some cases, the PCB 132, 232 couldbe used to heat the lid to a slightly higher temperature, e.g., up toabout 40° C. to reduce condensation on the lid 106, 206. In other cases,a positive pressure within the enclosure can be used to add a controlledleak across the lid 106, 206. The contour of the interior of the lid106, 206 could also be shaped to channel and control drainage of anycondensation formed on the lid 106, 206. The PCB 132, 232 can alsoinclude a conformal coating to reduce or minimize corrosion on the PCB132, 232 from condensation.

The incubators 100 as described herein may be further be understood byexamining the details in FIGS. 1-17. While FIGS. 1-17 illustrate variousfeatures of exemplary incubators, it is to be understood that theFigures are for illustration purposes only and in no way limit theinvention to the embodiment shown explicitly. Variations of each elementof the incubator 100 may be made as described throughout the descriptionherein.

FIGS. 1A-1B illustrate an isometric view and an exploded isometric viewof an incubator 100, respectively, in accordance with some embodiments.The incubator 100 includes an enclosure 102 where the enclosure 102includes a base 104 and lid 106. The base 104 and lid 106 can define theenclosure 102 having an internal chamber 110. The lid 106 includes aplurality of openings 112. The internal chamber 110 is sized to receivea cell culture plate 114. The incubator 100 includes a sealing element116 with a plurality of openings 118, which is illustrated with asealing element actuator 144. The illustrated cell culture plate 114includes a plurality of wells 120. The illustrated cell culture plate114 has an 8 by 12 arrangement of 96 wells 120. The openings 112 of thelid 106, openings 118 of the sealing element 116, and wells 120 of thecell culture plate 114 can be configured to be in register. Theincubator 100 includes a support 122 for the cell culture plate 114.

FIGS. 2A-2C illustrate a top view of a lid 106 and lid assembly 108 ofan incubator 100, the lid assembly 108 including printed circuit board(PCB) 132 and an optional spacer 134 respectively, in accordance withsome embodiments. FIGS. 3A-3C illustrate an exploded isometric view ofthe top sides of the lid 106, PCB 132 and spacer 134, respectively, thatcan be used in the embodiments of incubators 100 described herein. FIG.2A shows a top side 106A of the lid 106. The openings 112 of the lid 106extend through the thickness of the lid 106. The lid 106 also includes acut out 126 to accommodate another portion of the incubator 100, such asthe connector 136 of the printed circuit board 130. The lid 106 includesconnector openings 128 that can be used to removably attach the lid 106to the base 104. FIG. 2B shows a top view of a printed circuit board(PCB) 132 and connector 136. The PCB 132 includes openings 138. Theopenings 138 can be configured to be in register with the openings 112on the lid 106. Cutouts 130A can be used to place PCB 132 in registerwith the lid 106, allowing connectors to align connector openings 128 toattach the lid 106 and PCB 132 to the base 104. FIG. 2C shows a top viewof the spacer 134. The openings 142 can be configured to be in registerwith openings 112 on the lid and also with openings 138 on the PCB 132.Cutouts 130B of the spacer 134 can be used to place PCB 132 in registerwith the lid 106 and the PCB 132, allowing connectors to align connectoropenings 128 to attach the lid 106, PCB 132, and spacer 134 to the base104.

FIGS. 3D-3F illustrate an exploded isometric view showing the topsurface of a lid 106, a sealing element 116, and a printed circuit board132 and its associated connector, respectively, that can be used in theembodiments of incubators described herein. In contrast to FIGS. 3A-3C,the configuration illustrated in FIGS. 3D-3F omits the spacer 134 andpositions the sealing element 116 between the lid 106 and PCB 132.Positioning the sealing element 116 between the lid and PCB 132 canimprove the overall seal because the sealing element can form a sealbetween each of the lid 106 and PCB 132.

FIGS. 4A-B illustrate an exploded, isometric view of the bottom side ofthe lid 106 and PCB 132. In FIG. 4A, the bottom side 106B of the lid 106includes a recess 124 around a portion of a perimeter of the lid 106. Anupper side 132A of the PCB 132 (FIG. 3A) can be configured to engagewith the bottom side 106B of the lid 106. For example, the recess 124 ofthe lid 106 can be sized and shaped to receive the PCB 132. When the lid106 and PCB 132 are engaged, openings 112 and 138 may be aligned. Thenotch 126 may be sized to permit the connector 136 to fit when the lid106 and PCB 132 are engaged. Connector openings 128 may be used to alignthe lid 106 to the base 102. The openings 112 in the lid 106, may haveraised rings around the openings on the bottom side 106B of the lid 106.

As shown in FIG. 4B, the bottom side 132B of the PCB 132 is illustrated.Cutouts 130B can be used to align the PCB 132 when engaged with the lid106 as the lid 106 is attached to the base 104. A controller 174 (SeeFIG. 18) can control a heater 140 which is fabricated upon a bottomsurface 132B on the PCB 132. The heater 140 can be engaged with the PCB132 or integrally formed with the PCB 132 such that it does not obstructthe openings 138. The PCB 132 can include a plurality of sensors asdiscussed above, and can be present in any combination of temperature,humidity and/or gaseous phase sensors.

FIGS. 2A-C, 3A-F, and 4A-B taken together show various relationshipsbetween the lid, PCB 132 and optional spacer 134 for alignment and orderof mounting and for alignment of the openings in the lid and componentsof the lid assembly 108.

FIG. 5A illustrates a lid 206 that can be used in the embodiments ofincubators described herein. The lid 206 includes a lid cover 207. Theouter surface 207 of the lid 206 includes a marking 207 a of aninstruction such as “pull up to remove” on the lid cover 207. The lid206 also includes instructions 215 c on the compression tabs 215, suchas “push to install”. The marking 207 a and instructions 215 c cancolored, etched, or adapted to be machine readable by a computer imagingprogram.

The lid can include various protrusions and contouring on the undersideof the lid. FIG. 4A and FIG. 5B illustrate different lid configurations.FIG. 5B illustrates a view of a bottom surface of a lid 206 of anincubator in accordance with some embodiments. The lid 206 includes aplurality of openings 212. The lid 206 includes a patterned surface thatincludes a plurality of recesses that substantially surround groups 213of openings 212. The sealing element 116, 216 can move between theclosed and different open positions to allow a tool to access the wells120 of the cell culture plate 114. The groups 213 of the openings 212can improve a seal formed between the lid 206 and the sealing element116, 216 when the sealing element 116, 216 is in the closed position.For example, the openings 118, 218 in the sealing element 116, 216 canbe occluded by the space between the openings 212 of the groups 213. Theopenings 212 to the left side of the groups 213 can form a first subsetof openings while the openings 212 to the right side of the groups 213can form a second subset of openings 212.

FIGS. 5C-5E illustrate a top view of an incubator with a sealing elementat various positions in accordance with some embodiments. FIG. 5Cillustrates the sealing element 216 in a closed position such that theopenings 212 of the lid 206 are occluded. FIG. 5D illustrates thesealing element 216 in a first open position such that the openings 212of the lid 206 are open in every other row of openings 212 with theother rows of openings 212 occluded. FIG. 5E illustrates the sealingelement 216 in a second open position such that the openings 212 a ofthe lid 206 are open in every other row of openings 212 with the otherrows of openings 212 b occluded. The open openings 212 and occludedopenings 212 are reversed between the first open position (FIG. 12B) andsecond open position (FIG. 12C). In FIG. 5E the openings 212 b are openand 212 a are occluded. The lid 206 also includes four compression tabs215 with each tab having a surface 215 a adapted to engage a pin 215 bto secure the lid to the incubator. The compression tab 215 surface 215a can flex downward to engage the pin 215 b to provide a pressure tohold the lid 206 in place.

FIG. 5F-5G illustrate a top view of a sealing element 216 of anincubator at various positions in accordance with some embodiments. FIG.5F shows the sealing element 216 at a closed position such that theopenings 218 are not in register with openings 212 a/212 b of the lid206 (not shown). FIG. 5G shows the sealing element 216 at a first openposition such that the openings 218 are in register with a first subsetof openings 212 of the lid 206 (not shown). The illustrated sealingelement 216 has six rows of openings 218. The sealing element 216 can beused with the lids 206 illustrated in FIGS. 5C-5E with 12 rows ofopenings 212. The sealing element 216 can be moved between the closed,first open, and second open position depending on the desired well 120access for the cell culture plate 120. The illustrated sealing element216 can be positioned between the lid 206 and PCB 232. Positioning thesealing element 216 between the lid 206 and PCB 232 can improve theoverall seal because a seal can be formed between the sealing element216 and lid 206 and a second seal formed between the sealing element 216and PCB 232.

FIG. 5H illustrates a top view of an incubator including a printedcircuit board (PCB) 232 with openings 238 in accordance with someembodiments. The incubator is shown with the lid 206 and sealing element216 removed to expose the PCB 232. The PCB 232 can include a multi-layerconstruction. For example, in one embodiment the PCB 232 can include afour-layer board. Heating elements or devices, such as copper layers,can be internal layers of the PCB 232 to protect the heating elementsfrom exposure to moisture in the air within the enclosure. The use of amulti-layer construction for the PCB 232 can result in a thicker andless flexible PCB 232. The less flexible PCB 232 can improve the sealformed between the PCB 232 and the sealing element 216.

FIGS. 6A and 6B illustrate a top view of a portion of an incubator 100in accordance with some embodiments. The top view of the incubator 100shows the base 104 and sealing element 116. The sealing element 116includes sealing element openings 118. The sealing element 116 can bemoved with a sealing element actuator 144. Within the internal chamber110, the end of the cell culture plate support 122 distal to a frontside of the base 104 resting upon support notch 146 is visible. Aculture plate 114 is present in this view. Notches 148 in the upperinner edge of the base 104 permit movement of the sealing element 116 asit is actuated between a closed position and one or more open positions.Other arrangements of notches 148 are possible to support actuation tomore than one open position.

The sealing element 116 is in a first position in FIG. 6A and a secondretracted position in FIG. 6B. The sealing element actuator 144 isillustrated as rotating to retract the sealing element 116 in FIG. 6B.Movement of the sealing element 116 is facilitated by notches 148 on aninner upper edge of the base 104. The first position and secondretracted position can correspond to open and closed positions for thesealing element 116. In the open position the sealing element 116openings 118 line up with the wells 120 (not shown) of the cell cultureplate 114 (not labelled in FIG. 6B) and the lid openings 112 (not shownin this view) such that an import/export tip (not shown) can be used topass through the openings (112, 118) to access the wells 120 from theexterior of the incubator 100. In the closed position the sealingelement 116 blocks or partially obscures the openings 112 of the lid 106(not shown in this view). It is not required that sealing element 116form an air tight seal for the openings 112 of the lid 106. For example,in some cases the sealing element 116 can allow a purge gas to flow outof the interior chamber 110 of the incubator 100.

FIGS. 7-8 illustrate an exploded isometric view of a portion of anincubator 100, showing the sealing element 116 having openings 118connected to actuator 144; a cell culture plate 114 with wells 120; andbase 104 with an access door 154 in a closed and an open position,respectively. Visible in the view of the base 104 in FIGS. 7 and 8 isinternal chamber 110, support notch 146 upon which cell culture platesupport 122 rests when the incubator 100 is in the closed position.Support notch 146 may be formed in the inner surface of the base 104within the enclosure 102. Passages 150A and 150B are connected throughthe side of the base 104 for gaseous input and fluid drainage,respectively. The passages 150A and 150B can be sealable. Notches 148are formed in the upper inner surface of the base 104 for movement ofthe sealing element 116. Electrical connections 152 are shown connectingto the side of the base 104. The cell culture plate support 122 andaccess door 154 can form an access assembly 168 (FIG. 8). Access door154 may have a handle 172. The access assembly 168 can also include afront plate 156 that sealably interfaces with a front portion 158 of theenclosure 102 of the incubator 100. The access assembly 168 may alsoinclude access track guides 166 to support the movement of the cellculture plate 114 and support in and out of the enclosure 102. Theillustrated access assembly 168 has the cell culture plate support 122mounted on the front plate 156 of the access door 154. Insulation panels170 may be attached to the base 104.

The assembly 164 can be mounted on an enclosure support 160 thatsupports the enclosure 102. The enclosure support 160 can include tracks162 that can allow the access assembly 168 to slide on the track guides166 relative to the enclosure 102 between a closed position (FIG. 7) andan open position (FIGS. 8 and 9). The enclosure support 160 can alsoinclude legs 164 to further support the base 104, which may beadjustable.

FIG. 9 illustrates a top view of the incubator 100 and access door 154in the open position. In this illustration, the extent of the tracks 162is visible as tracks 162 extend beyond a rear side of the base 104,permitting the track guides 166 to fully slide and close the cellculture plate support 122 to rest within support notch 146. The tracks162 are mounted on enclosure support 160. The cell culture plate support122 is attached to front plate 156 of the access door 154, formingaccess assembly 168, and may further include track guides 166. Accessdoor 154 may have a handle 172. Sealing element 116, attached toactuator 144 is located upon the base 104, fitting within notches 148which permit the sealing element 116 to move when actuated. Within theinternal chamber 110, support notch 146 in the base 104 is shown whenthe cell culture plate support 122 is not engaged (i.e. the accessassembly is open). Notches 148 are shown in the upper inner surface ofthe base 104, which permit the sealing element 116 to move whenactuated. Insulation panels 170 may be attached to the base 104.

FIGS. 10A-10B illustrate a top view of an incubator with a support 222for a cell culture plate in an open and a closed position, respectively,in accordance with some embodiments. FIG. 10A illustrates the support222 with a different shape than the support 122. The support 222 can bemoved by grabbing the handle 272 to slide the rails 266 to the openposition shown in FIG. 10B. The support 222 can be positioned near thebottom of the enclosure to allow for air-space above the cell cultureplate 114. Allowing air-space above the cell culture plate 114 can beimportant for maintaining a proper humidity level above the wells 120 ofthe cell culture plate 114 and avoiding evaporation of the culturemedium contained within the wells 120 of the cell culture plate 114. Thesupport 222 is attached to the access door 254 such that sliding theaccess door horizontally also moves the support 222 as shown in FIG.10B. The support 222 can be attached directly or indirectly to accessdoor 254. FIG. 10B shows the rails 266 with a cylindrical shape. Therails 266 can slide along a complimentary shaped opening in theenclosure support 260.

FIGS. 11A-11B illustrate views of a portion of a support 122 for a cellculture plate of an incubator in accordance with some embodiments. Thesupport 122 is part of the access assembly 268. The access assembly 268includes four openings 269 configured to hold vials or test tubes in theaccess door 254. The access assembly 268 includes a front plate 256. Theaccess door 254 has a handle 272. The illustrated front plate 256 has afloating engagement with the access door 254 via the biased connections255. The front plate 256 is directly attached to the support 222. Thebiased connections 255 are illustrated with a screw attached to thefront plate 256 and a spring surrounding each of the screws to bias orpress the front plate 256 relative to the access door 254. When theaccess assembly 268 is in the open position, the heads of the screws areretained on the C-bores of the access door 254. When the access assembly268 is in the closed position the biased connections 255 can provide aforce to the front plate 256 to secure the front plate 256 relative tothe enclosure.

FIG. 11C illustrates a side view of an incubator in accordance with someembodiments. FIG. 11C illustrates the access assembly 268 in an almostclosed position. FIG. 11C shows the biased connections 255 securing thefront plate 256 against the enclosure. The rails 266 can be securedagainst a complementary connection 267, such as a flexible locking pin,within the enclosure support 260 in the closed position. FIG. 11Cillustrates the front plate 256 against the enclosure and the rails 266just prior to engagement of the complimentary connection 267 prior toclicking into the closed position. The biased connections 255 can alsoreduce the amount of abrupt motion experience by the support 222 andthus any well-plate supported by the support 222 as the access door 268is opened or closed and locked into place. Reducing the abrupt motioncan minimize and prevent splashing and sloshing of any fluid in thewells 120 of the cell culture plate 114 when the access door 254 isclosed. In order to close the illustrated incubator, the rails accessassembly 268 is advanced until the front plate 256 engages with theenclosure. Next, an additional force can be applied to further move theaccess assembly 268 and the rails 266 such that the rails are securedagainst a complementary connection 267 within the enclosure support 260to enter the closed position. The biased connections 255 reduce oreliminate movement of the cell culture plate 120 caused by the force toadvance the access assembly 268 after the front plate 256 is engagedwith the enclosure to enter the closed position.

The rails of the access assembly 168, 268 can include one or more stopsor engagement surfaces to help hold the access assembly 168, 268 in oneor more discrete positions such as open and closed position. FIGS. 12Aand 12B illustrate a view of a magnet and a view of rails that can beused in embodiments of the incubators disclosed herein. A magnetassembly 295 is shown with a magnet 297 and a magnet housing 299. Themagnet assembly 295 can be included within the rails 266. The magnet 297within the magnet assembly 295 can engage with a complementary magnetwithin the enclosure support 260 to form a connection to hold the rails266 in place at a designed location, such as the open or closedpositions for the access assembly 268. In some embodiments the magnet297 can engage with the door switch 273 (FIG. 16). FIG. 13 illustratesan embodiment of rails 266 that can be used in the embodiments ofincubators described herein. FIG. 13 shows rails 266 with flat surfaces266 a that are designed to interface with a complementary connection267, such as a flexible locking pin, within the enclosure support 260.The locking pins help to hold the access assembly 268 in open or closedpositions.

FIG. 14 illustrates an exploded view of a portion of an incubatorincluding an enclosure support 260 in accordance with some embodiments.The illustrated enclosure support 260 includes a heat transfer element261. The heat transfer element 261 can provide heat to the bottom of theenclosure. The heat transfer element can be in contact with a circulatedheating/cooling fluid, resistive heater or other heating/cooling device.The illustrated enclosure support 260 includes a gasket material 263.The gasket material 263 can prevent condensation or liquid from fallingdown into the enclosure support and contacting any of the electroniccomponents with the enclosure support 260. The enclosure support canoptionally include a drip tray or other drain to channel condensationand other liquid along a desired path to avoid or minimize condensationfrom contacting the electronic components. The enclosure support haselectrical connections 252 that may be connected to heat transferelement 261 and other electronics in the incubator.

FIG. 15 illustrates an exterior portion of an incubator in accordancewith some embodiments. The enclosure support 260 can include tracks 262that can allow the access assembly 268 to slide on the rails 266relative to the enclosure between a closed position and an openposition. The enclosure support 260 includes a heat exchange fluid inlet265 a and outlet 265 b. The enclosure support 260 includes differentelectrical connection ports 271 a, 271 b, and 271 c. For example,electrical connection port 271 a is illustrated as an Ethernet port. Theelectrical connection ports 271 a, 271 b, and 271 c can be used tocontrol, monitor, and update software/firmware of the incubator.

FIG. 16 illustrates a side view of an incubator in accordance with someembodiments. The enclosure support 260 and access assembly 268 isillustrated in a closed position. The enclosure support 260 includes adoor switch 273 that can mechanically, electrically, or magneticallyengage with a portion of the rail 268 when the access assembly 268 is inthe closed position. The door switch 273 can recognize when the accessassembly 268 is in a closed position and transmit that information to aprocessor onboard the incubator.

FIGS. 17A-17B illustrate an isometric view of an incubator with asupport 222 for a cell culture plate in an open and a closed position,respectively, in accordance with some embodiments. The illustratedsupport 222 includes a distal lip 223 configured to engage an edge ofthe cell culture plate 114. The distal lip 223 can be supported by theresting support 246 when the support 222 is in the closed position. Thedistal lip 223 of the support 222 can also rest against a portion 247 ofthe enclosure as shown in FIG. 23A when in the open position to preventvibration and motion of the support.

In FIGS. 1, 3, and 6-9, the figures show the sealing element 116, 216having 96 openings 118, 218 which may be moved into register with 96wells 120 of the cell culture plate 114, but other configurations arealso envisioned. In some embodiments there may be a first set ofopenings 118, 218 that may have 96 openings, plus a second set ofopenings 118, 218 that may be moved into register with fewer than 96 ofthe wells 120 on the cell culture plate 114. As non-limiting examples,the second set may be moved to register with half of the 96 wells 120 ormay be moved to be in register with 24 of the 96 wells 120. In someembodiments, there may further be a third set of openings 118, 218 onthe sealing element 116, 216 which may be moved into register with fewerthan all 96 wells 120 of the cell culture plate 114. As non-limitingexamples, the third set of openings 118, 218 may be 48 openings that maybe moved into register with half of the wells 120 or may be 24 openingsthat may be moved into register with a quarter of the wells 120. Thethird set of openings 118, 218 may move into register with wells 120that are different from the wells 120 that may be accessed by use of thesecond set of openings 118, 218. When the second and third set ofopenings 118, 218 provide access to different wells 120, the wells 120may be located on different halves or sides of the cell culture plate114, may be physically alternating in location or may be locatedaccording to another preselected pattern.

In other embodiments, the first set of openings 118, 218 may be movedinto register with fewer than all 96 wells 120 of the cell culture plate114. Non-limiting examples include where the first set of openings 118,218 may move into register with half or a quarter of the wells 120 ofthe cell culture plate 114. The sealing element 116, 216 may furtherhave a second set of openings 118, 218 that may be moved into registerwith fewer than 96 of the wells 120 on the cell culture plate 114. Asnon-limiting examples, the second set may be moved to register with halfof the 96 wells 120 or may be moved to be in register with 24 of the 96wells 120. In some embodiments, there may further be a third set ofopenings 118, 218 on the sealing element 116, 216 which may be movedinto register with fewer than all 96 wells 120 of the cell culture plate114. As non-limiting examples, the third set of openings 118, 218 may be48 openings that may be moved into register with half of the wells 120,or may be 24 openings that may be moved into register with a quarter ofthe wells 120. The first, second or third sets of openings 118, 218 maymove into register with wells 120 that are different from the wells 120that may be accessed by use of either of the other two sets of openings118, 218, or may access overlapping locations of wells 120. The wells120 may be located on different halves or sides of the cell cultureplate 114, may be physically alternating in location or may be locatedaccording to another preselected pattern.

While FIGS. 1 and 7-9 illustrate a cell culture plate 114 having 96wells 120, it is also envisioned that the lid 106, 206, sealing element116, 216, PCB 132, 232, spacer 134 and the respective openings (112,212, 138, 238, and optionally 142) thereof, can accommodate differentlyproportioned cell culture plates 114 and/or cell culture plates 114having different numbers of wells 120 within them. In some embodiments,there may be 384 wells in the cell culture plate 114. When 384 wells 120are present, the lid 106, 206 and lid assembly 108 components includingthe PCB 132, 232 and optional spacer 134 may have 384 openings or somesubset thereof. When the cell culture plate 114 has 384 openings thesealing element 116, 216 may have 384 openings 118, 218 that can bemoved into register with the wells 120, or may have fewer openings 118,218 that can be moved into register with a subset of the wells 120. Thesealing element 116, 216 may have additional sets of openings 118, 218that may be configured as described above for a 96 cell culture plate114 configuration, and may be configured in any similar combination. Thecell culture plate 114 may also be configured to have 12 or 6 or fewerwells 120, and the lid 106, 206, sealing element 116, 216. PCB 132, 232,optional spacer 134 and the respective openings (112, 212, 138, 138, andoptionally 142) thereof, may be configured to provide access to thissmaller number of wells 120 and/or subsets thereof.

Any of the incubators 100 described above may have any suitablecombination of sizes of openings 112, 212, 138, 238, and/or 142, and/orany of the additional components such as insulation, sensors, one ormore controllers 174 (See FIG. 18), electrical connections 152, 252heating and cooling devices, inlets for gas and for fluid drainage inany combination. The one or more controllers 174 may control the sealingelement 116, 216, the temperature, relative humidity, and/or gaseousenvironment of the internal chamber 110, and/or the access assembly 168,268.

In some embodiments, an incubator 100 includes an enclosure 102 havingan internal chamber 110 configured to support a cell culture plate 114comprising a plurality of wells 120, wherein the enclosure 102 has aplurality of openings provided by the openings 112 in the lid 106, 206and openings (138, 238 and optionally 142) of the associated lidassembly 108. The openings in the enclosure (112, 212, 138, 238 andoptionally 142) are configured to allow access to the wells 120 of thecell culture plate 114; and a sealing element 116, 216 configured toseal the plurality of openings in the enclosure 102, wherein the sealingelement 116, 216 includes a first plurality of openings 118, 218corresponding to at least a subset of the plurality of openings in theenclosure. The enclosure 102 may include a base 104 and a lid 106 wherethe base 104 and the lid 106 defines the internal chamber. In someembodiments, the internal chamber 110 has a volume of about 200 cm³ toabout 750 cm³. In other embodiments, the internal chamber 110 has avolume of about 400 cm³ to about 1,000 cm³. The base 104 may be formedfrom a rigid material having a high thermal conductivity and low thermalcapacitance. The lid 106 may be formed from an insulating plastic. Theincubator 100 may include a printed circuit board (PCB) 132, 232. ThePCB 132, 232 may be located between the sealing element 116, 216 and aninternal surface of a top of the enclosure. PCB 132, 232 may include aplurality of openings 138, 238 in register with the plurality ofopenings passing through the enclosure. The PCB 132, 232 may include oneor more sensors, which may be selected from the group consisting of: atemperature sensor, a humidity sensor, an oxygen sensor, and a carbondioxide sensor. The incubator 100 may include a spacer 134, wherein thespacer 134 is located between the PCB 132, 232 and the sealing element116. The spacer may 134 include a plurality of openings 142 in registerwith the plurality of openings passing through the enclosure and withthe plurality of openings 138, 238 of the PCB. The spacer 134 may beconfigured to engage with the sealing element 116. In some embodiments,the incubator 100 has no spacer 134. The sealing element 116, 216 of theincubator 100 may be movable between a closed position where the sealingelement 116, 216 occludes each of the plurality of openings in theenclosure and a first open position where the first plurality ofopenings 142 of the sealing element 116, 216 are in register with the atleast a subset of the plurality of openings in the enclosure 102. Thefirst plurality of openings 138, 238 of the sealing element 116, 216 maybe the same as the number of openings in the enclosure 102. In someembodiments, the sealing element 116 has only a first plurality ofopenings 138. In some embodiments, the enclosure 102 and the sealingelement 116, 216 have 96 openings 138. In other embodiments, theenclosure 102 and the sealing element 116, 216 have 384 openings. Thesealing element 116, 216 may further include a second plurality ofopenings 138, 238, the second plurality of openings 138, 238 beingdifferent from the first plurality of openings 138, 238. The number ofopenings 138, 238 in the second plurality of openings 138, 238 in thesealing element 116, 216 may be one-half, one-third, or one-fourth thenumber of openings in the enclosure 102. Each opening of the pluralityof openings in the enclosure may have a diameter of about 1 mm to about10 mm or about 1 mm to about 5 mm. Each opening of the plurality ofopenings 138, 238 in the sealing element 116, 216 may have a diameter ofabout 1 mm to about 10 mm or about 1 mm to about 5 mm. The incubator 100may include a first heating/cooling device engaged with the enclosure,the first heating/cooling device controlled by a temperature controllerattached to the incubator. The first heating/cooling device may beselected from the group consisting of: a resistive heater, a fluid coilconfigured to circulate a heat exchange fluid, and one or more Peltierdevices. The first heating/cooling device may directly contact an outersurface of the bottom of the enclosure. The first heating/cooling devicemay include a fluid coil. The incubator may include a secondheating/cooling device, which may be located within the enclosure. Thesecond heating/cooling device may be engaged with a top of the enclosureand may be controlled by a temperature controller attached to theincubator. The second heating/cooling device may be resistive heatingelements 140 that are part of the PCB 132, and are located on a side ofthe PCB 132 facing the internal chamber of the enclosure. The secondheating/cooling device may include a plurality of openings that are inregister with the plurality of openings in the enclosure. The incubator100 may include a controller 174, which may be a temperature controllerconfigured to maintain a temperature of the internal chamber within adesired range by controlling the first and/or second heating/coolingdevice. The controller 174 may also control the sealing element 116, 216relative humidity, gaseous environment of the internal chamber, and/orthe access assembly 168. The incubator 100 may include a support 122,222 for the cell culture plate 114. The support 122, 222 may beconfigured to slideably move relative to the enclosure 102 from aposition within the enclosure 102 to a position outside of the internalchamber 110 of the enclosure 102. The incubator 100 may further includean access door 154, 254 attached to the support 122, 222 for the cellculture plate 114. The support 122, 222 and access door 154, 254 mayform an access assembly 168, 268, including a front plate 156, 256 thatsealably interfaces with a portion of the enclosure. The access assembly168, 268 may be movably mounted on an enclosure support 160, 260 thatsupports the enclosure 102. The incubator 102 may further include atleast one passage 150A in the enclosure configured for gas, where the atleast one passage 150A may be located on a wall of the base 104 at thesame height from a bottom of the base as the side of the cell cultureplate 114.

Methods.

Methods are also provided for using the incubators 100 disclosed herein.The methods can include moving a sealing element 116, 216 with aplurality of openings 118, 218 to an open position where the pluralityof openings 118, 218 of the sealing element 116, 216 are in registerwith a first subset of openings of the plurality of openings (112, 212,138, 238 and optionally 142) in the enclosure 102, as provided by thelid 106, 206 and associated lid assembly 108. The plurality of openings118, 218 of the sealing element 116, 216 and the first subset ofopenings of the plurality of openings (112, 212, 138, 238 and optionally142) in the enclosure 102 provide a first plurality of passages from anexterior of the incubator 100 to an internal chamber 110 of theenclosure 102. An import/export tip can be advanced through one or moreof the first plurality of passages between the exterior of the incubator100 and the internal chamber 110 of the enclosure 102. The methods caninclude collecting or depositing a material within the internal chamber110 of the enclosure 102 via the import/export tip. The first subset ofopenings can include all of the openings of the plurality of openings inthe enclosure, or less than all (e.g., ½, ⅓, ¼, or less).

Material that may be collected from, withdrawn from, or deposited to awell 120 of a cell culture plate 114 within the internal chamber 110 ofthe enclosure 102 may include micro-objects, (which may further includeone or more biological micro-objects), proteins, nucleic acids, lipidsor other cellular components found within or secreted by biologicalmicro-objects, fluids such as but not limited to culture media, solventssuch as but not limited to dimethylsulfoxide or ethyl alcohol,surfactants, assay reagents, or reagents such as permeabilizationreagents, labelling reagents, fusion reagents, and the like, and thewaste products derived from culturing or reaction of the reagents with acomponent of the material being withdrawn or deposited. In variousembodiments, the material may contain at least one biological cell whichmay be maintained or expanded in the well 120 of the culture plate 114.In other embodiments, the material may not have a cell present but maycontain the derived proteins, nucleic acids, lipids or other cellularcomponents described above which may be suitable for holding underprescribed temperature and/or humidity conditions. In yet otherembodiments, the material that is deposited to a well may be one or morereagents to assay, fix, transfect or stabilize biological micro-objectsor components found within or secreted by the biological micro-objects.In yet other embodiments, the material that is deposited to or withdrawnfrom a well 120 can include a micro-object such as a bead, and the like.The bead may include a protein, saccharide, and/or a label (where thelabel may be detected colorimetrically, fluorescently, orluminescently). In some embodiments, the material may include more thanone type of material described above.

Collecting or depositing the material includes collecting or depositingthe material within a well 120 of a cell culture plate 114 within theinternal chamber 110 of the enclosure 102. In some embodimentscollecting or depositing the material can be done with an import/exporttip. In some embodiments the import/export tip can include a pluralityof tips. In some embodiments the plurality of tips of the import/exporttip can simultaneously collect or deposit the material from a pluralityof the wells 120 of the cell culture plate 114 within the incubator 100.The import/export tip(s) can be withdrawn through the one or more of thepassages between the exterior of the incubator 100 and the internalchamber 110 of the enclosure 102 after collecting or depositing thematerial. Collecting or depositing the material can be performedrobotically. In various embodiments, the import/export tip maywithdraw/deposit material at a rate of about 0.01 μl, 0.02 μl, 0.05 μl,0.1 μl, 0.2 μl, 0.5 μl, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl,9 μl, 10 μl, 11 μl, 12 μl, 15 μl, 17 μl, 20 μl, 22 μl, 24 μl, 25 μl, 27μl, 29 μl, 30 μl per sec, or any range defined by two of the foregoingvalues.

Methods are also provided for agitating the contents of a well 120 inthe cell culture plate 114 in conjunction with collecting or depositingthe material in the well 120. In some embodiments, a mixing tip isinserted through one or more of the passages between the exterior of theincubator 100 and the internal chamber 110 of the enclosure 102. Thepassages are created by bringing an opening 118, 218 of the sealingelement 116, 216 into register with an opening (112, 212, 138, 238 andoptionally 142) in the enclosure 102 of the incubator 100. The mixingtip may provide agitation within fluid present in the well 120 byrotating, vibrating, or otherwise moving about, injecting fluid (such asculture medium), injecting gas, or the like. The agitation may providemore uniform samples of the material present in the well or may providemore uniform composition of a liquid medium within a well prior toadding material to it, or prior to adding another composition containingeither a material of same or differing type (e.g. a biologicalmicro-object of a different type or a micro-object such as a bead havinga label or a reagent bound to it) or other chemical components that maybe desired. In some embodiments, the mixing tip may withdraw an aliquotof fluid from the well 120 and reinject it to mix the contents of thewell 120 before material is added to the well 120 or is withdrawn fromthe well 120 by the import/export tip (not shown). In some embodiments,the mixing tip may withdraw about 10 μl to about 50 μl of fluid from thewell and reinject it to the well at a rate of about 1 μl, 2 μl, 3 μl, 4μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 15 μl, 17 μl, 20μl, 22 μl, 24 μl, 25 μl, 27 μl, 29 μl, or about 30 μl per sec. In someembodiments, the mixing tip withdraws about 10 μl, 11 μl, 12 μl, 13 μl,14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, 21 μl, 22 μl, 23 μl, 24μl, 25 μl, 26 μl, 27 μl, 28 μl, 29 μl, 30 μl, 35 μl, 40 μl, 45 μl, orabout 50 μl of fluid to mix the contents of the well before withdrawingfrom or depositing to a well 120.

The methods also provide for cleaning steps before and/or after each useof the import/export tip and/or the mixing tip. The cleaning step caninclude manual wiping with a tissue or cloth, a water/bleach flush overthe tip, water/bleach dip, ultrasonic cleaning or dip into ozonizedwater.

The sealing element 116, 216 can be moved to a closed position such thatthe sealing element 116, 216 occludes the plurality of openings (112,212, 138, 238 and optionally 142) in the enclosure 102. Moving thesealing element 116, 216 between the open position and closed positioncan include sliding the sealing element 116, 216 relative to theenclosure 102. The sealing element 116, 216 can be moved between theopen and closed positions by actuating a sealing element actuator 144.For example, a motor or rotary solenoid can be used to actuate thesealing element actuator 144. The sealing element 116, 216 can be in theopen position for an amount of time which is sufficiently short so as toprevent a carbon dioxide content and/or a humidity of air present in theinternal chamber 110 from equilibrating with a carbon dioxide contentand/or a humidity of air surrounding the incubator 100.

Methods are also provided for moving the sealing element 116, 216between the closed position and a plurality of open positions. Themethods can include moving the sealing element 116, 216 to a first openposition in which a first plurality of openings 118, 218 in the sealingelement 116, 216 are in register with a first subset of the plurality ofopenings (112, 212, 138, 238 and optionally 142) in the enclosure 102.In some embodiments, the number of the plurality of openings 118, 218 inthe sealing element 116, 216 can be the same as a number of theplurality of wells 120 in the cell culture plate 114. In someembodiments, a number of openings 118, 216 in the first plurality ofopenings 118, 218 in the sealing element 116, 216 is equal to or lessthan one-half, one-third, one-fourth, one-sixth, or one-twelfth a numberof the plurality of wells 120 in the cell culture plate 114.

The methods can include moving the sealing element 116, 216 to a secondopen position in which a second plurality of openings 118, 218 in thesealing element 116, 216 are in register with a second subset of theplurality of openings (112, 212, 138, 238 and optionally 142) in theenclosure 102 and all other openings of the plurality of openings (112,212, 138, 238 and optionally 142) in the enclosure 102 are occluded. Thesecond plurality of openings 118, 218 in the sealing element 116, 216and the second subset of the plurality of openings (112, 212, 138, 238and optionally 142) in the enclosure 102 can provide a second pluralityof passages from an exterior of the incubator 100 to the internalchamber 110. In various embodiments, a number of openings 118, 218 inthe second plurality of openings 118, 218 in the sealing element 116,216 is equal to or less than one-half, one-third, one-fourth, one-sixth,or one-twelfth a number of the plurality of wells 120 in the cellculture plate 114.

The methods can also include moving the sealing element 116, 216 to athird open position in which a third plurality of openings 118, 218 inthe sealing element 116, 216 are in register with a third plurality ofopenings (112, 212, 138, 238 and optionally 142) in the enclosure 102and all other openings of the plurality of openings (112, 212, 138, 238and optionally 142) in the enclosure 102 are occluded. The thirdplurality of openings 118, 218 in the sealing element 116, 216 and thethird subset of the plurality of openings (112, 212, 138, 238 andoptionally 142) in the enclosure 102 can provide a third plurality ofpassages from an exterior of the incubator 100 to the internal chamber110. In some embodiments, a number of openings in the third plurality ofopenings 118, 218 in the sealing element 116, 216 is equal to or lessthan one-half, one-third, one-fourth, one-sixth, or one-twelfth a numberof the plurality of wells 120 in the cell culture plate 114. In variousembodiments of the method, the first plurality of the openings 118, 218in the sealing element 116, 216, the second plurality of openings 118,218 in the sealing element 116, 216 and the third plurality of openings118, 218 in the sealing element 116, 216, if present, arenon-overlapping. In some embodiments first plurality of the openings118, 218 in the sealing element 116, 216, the second plurality ofopenings 118, 218 in the sealing element 116, 216, and the thirdplurality of openings 118, 218 in the sealing element 116, 216, ifpresent, when in register with the openings (112, 212, 138, 238 andoptionally 142) in the enclosure 102, provide access to wells 120 indifferent portions of the cell culture plate 114. In some embodiments,the actuator 144 may move the sealing element 116, 216 from the closedposition to the first open position and from the closed position to thesecond open position. In some embodiments, the actuator 144 may furthermove the sealing element 116, 216 from the closed position to the openposition. In other embodiments, the actuator 144 may move the sealingelement 116, 216 from the closed position to any of the first openposition, second open position and/or the third open position, ifpresent.

The methods can also include measuring one or more of a temperature, ahumidity, and a carbon dioxide content of the internal chamber 110 ofthe enclosure 102 and controlling one or more of the temperature,humidity, and carbon dioxide content of the internal chamber 110 of theenclosure 102. Controlling the temperature can include heating orcooling the internal chamber 110 of the enclosure 102. Controlling thehumidity can include providing a humidity source to the internal chamber110 of the enclosure 102. Controlling the carbon dioxide content caninclude providing a carbon dioxide source to the internal chamber 110 ofthe enclosure 102.

In some embodiments, the pressure of the internal chamber 110 of theincubator 100 can be maintained at a desired range while the sealingelement 116 is in the closed position. The sealing element 116, when inthe closed position, can be capable of maintaining a pressure within theinternal chamber 110 of the enclosure 102 between about 0.0005 psi toabout 0.0100 psi above ambient pressure or any of the pressure rangesdisclosed herein. For example, the pressure of the internal chamber 110can be maintained between about 0.0005 psi to about 0.01000 psi aboveambient pressure. In some embodiments, the pressure of the internalchamber 110 may be maintained about 0.0005 psi, 0.0010 psi, 0.0015 psi,0.0020 psi, 0.0025 psi, 0.0030 psi, 0.0035 psi, 0.0040 psi, 0.0045 psi,0.0050 psi, 0.0055 psi, 0.0060 psi, 0.0065 psi, 0.0070 psi, 0.0075 psi,0.0080 psi, 0.0085 psi, 0.0090 psi, 0.0095 psi, or about 0.0010 psi.Clean rooms typically use a positive pressure of about 0.0072 psi orless. In some embodiments the pressure of the internal chamber 110 canbe maintained less than about 0.0072 psi above ambient pressure. In someembodiments the pressure of the internal chamber 110 can be maintainedabove about 0.0072 psi above the ambient pressure.

A purge gas can be provided to maintain the pressure in someembodiments. The methods can include providing the purge gas to theinternal chamber 110 of the enclosure 102 whereby, when the sealingelement 116, 216 is in the closed position and the support 122, 222 forthe cell culture plate 114 is positioned inside the internal chamber 110of the enclosure 102, the pressure within the internal chamber 110 ofthe enclosure 102 is maintained between about 0.0005 psi to about 0.0100psi above ambient pressure. The purge gas can include one or more ofcarbon dioxide, oxygen, nitrogen, and noble gases. In some embodiments,the purge gas may include about 5% carbon dioxide by volume.

In some embodiments the pressure can be maintained with the purge gassuch that a desired flow rate of purge gas is achieved through theopenings. In some cases, the flow rate can be less than or about 10liters per hour, 9 liters per hour, 8 liters per hour, 7 liters perhour, 6 liters per hour, 5 liters per hour, 4 liters per hour, 3 litersper hour, 2 liters per hour, 1 liter per hour, or any range defined bytwo of the foregoing values. The flow rate may be more than about 0.5liter per hour. In some embodiments the flow rate can be about 1liter/hour to about 10 liters/hour.

In some embodiments a positive pressure can be maintained within theinternal chamber 110 when the sealing element 116, 216 is in the openposition. For example, the purge gas can be provided when the sealingelement 116, 216 is in the open position to decrease the likelihood ofcontamination.

The cell culture plate 114 can be provided to the incubator 100 bysliding the support to withdraw the support from the internal chamber110 of the enclosure 102 to a position outside of the internal chamber110 of the enclosure 102 followed by placing the cell culture plate 114on the support while the support is in the position outside of theinternal chamber 110 of the enclosure 102. Placing the cell cultureplate 114 can done by a human operator or a robotic tool. After placingthe cell culture plate 114 on the support 122, 222, the support 122, 222can slide to a position inside the internal chamber 110 of the enclosure102 and thereby moving the cell culture plate into the internal chamber110 of the enclosure 102. Sliding the support 122, 222 can includesliding an access door 154, 254 attached to the support for the cellculture plate 114. Sliding the support 122, 222 can include sliding thesupport along one or more tracks 162, 262 on an enclosure support 160,260 of the incubator 100. Sliding the support 122, 222 can be done by ahuman operator or a robotic tool. After loading the cell culture plate114 within the enclosure 102 an environment within the internal chamber110 of the enclosure 102 can be established to support a materialsupported by the cell culture plate 114.

The cell culture plate 114 can be removed from the support for the cellculture plate 114 similarly to the loading steps described above. Thesupport 122, 222 for the cell culture plate 114 can be accessed bysliding the support 122, 222 from the internal chamber 110 of theenclosure 102 to the position outside of the internal chamber 110 of theenclosure 102 and thereby withdrawing the cell culture plate 114 fromthe internal chamber 110 of the enclosure 102. Sliding the support 122,222 can be done by sliding an access door 154, 254 attached to thesupport 122, 222. In some embodiments sliding the support 122, 222 canbe done by a human operator. In some embodiments sliding the support122, 222 can be done robotically, such as by a robotic tool. After thecell culture plate 114 is at the position outside of the internalchamber 110 of the incubator 100 the cell culture plate 114 can beremoved from the support. Removal can be done by a human operator or arobotic tool.

In various embodiments, methods are provided to deliver one or moresamples containing material to one or more wells 120 of the cell cultureplate 114 contained within the well plate incubator 100, where thesamples may be obtained from a macroscale cell culture apparatus, amicrofluidic device and/or an analytical instrument. In someembodiments, the material may include a biological micro-object capableof being maintained and/or expanded. The sample containing thebiological micro-object may be provided such that the biologicalmicro-object is isolated away from other biological micro-objects thatmay be different from the biological micro-object or may be simplyselected to be a single representative of a desired set of biologicalmicro-objects present in the macroscale cell culture apparatus,microfluidic device and/or analytical instrument. In some embodiments, asingle biological micro-object delivered within the material may beexpanded to form a clonal population within the continuous access cellculture incubator. In some embodiments, a sample obtained from amacroscale cell culture apparatus may provide samples having biologicalmicro-objects which may or may not be already sorted. The microfluidicdevice and/or analytical instrument from which samples are obtained maysort biological micro-objects, may provide dissociated biologicalmicro-objects, or may have provided chemical or other treatment to thebiological micro-object(s) delivered to the cell culture plate, to namesome non-limiting exemplars.

Material may be delivered to a well 120 of the cell culture plate 114that already contains media or other reagents. In some embodiments, thecell culture plate 114 of the continuous access incubator 100 maycontain reagents to provide a treatment to a cell delivered to a welltherein. For example, lysis reagents may be present or a fluidic mediummay be present which will prepare a cell for further processing such asfreezing, lysis or permeabilization.

In other embodiments, methods are provided to deliver a samplecontaining a biological micro-object withdrawn from a well in the cellculture plate within the continuous access incubator to a macroscalecell culture apparatus, a microfluidic device (which may be ananofluidic device), an analytical instrument, or to a storage device.The biological micro-object may have been cultured (i.e. grown undersuitable conditions) for a preselected period of time before beingwithdrawn for delivery to a macroscale cell culture apparatus, amicrofluidic device or an analytical instrument. In other embodiments,the biological micro-object may be treated while present in thecontinuous access incubator to permeabilize a cell present in thebiological micro-object or may be lysed for further analysis in amicrofluidic device or analytical instrument, to name two non-limitingexamples. In some embodiments, the biological micro-object may betreated to be stabilized for analysis or for storage. One non-limitingexample includes treating the biological micro-object with suitablemedia to stabilize for freezing and long term storage.

In some embodiments methods for accessing an internal chamber 110 of anincubator 100 are provided. The incubator 100 can include an enclosure102 having a plurality of openings and a sealing element 116, 216 havingmore than one plurality of openings 118, 218. Each plurality of openings118, 218 in the sealing element 116, 216 can correspond to at least asubset of the plurality of openings in the enclosure 102. The methodscan include moving the sealing element 116, 216 to a first open positionand thereby bringing a first plurality of openings 118, 218 in thesealing element 116, 216 into register with a first subset of theplurality of openings in the enclosure 102. The first plurality ofopenings 118, 218 in the sealing element 116, 216 and the first subsetof openings in the plurality of openings in the enclosure providing,when in register, a first plurality of passages from an exterior of theincubator 100 to the internal chamber 110 of the enclosure 102. Themethods can include advancing an import/export tip through one or moreof the first plurality of passages between the exterior of the incubator100 and the internal chamber 110 of the enclosure 102. The methods caninclude collecting or depositing a material with the import/export tipwithin the internal chamber 110 of the enclosure 102. The methods canfurther include moving the sealing element 116, 216 to a closedposition, and thereby occluding each of the plurality of the openings inthe enclosure 102.

When the sealing element 116, 216 is in the open position, the firstplurality of openings 118, 218 in the sealing element 116, 216 can beconfigured to be in register with a first subset of a plurality of wells120 in the cell culture plate 114. In some embodiments a number of theplurality of openings 118, 218 in the sealing element 116, 216 is thesame as a number of the plurality of wells 120 in the cell culture plate114. In some embodiments a number of the plurality of openings 118, 218in the sealing element 116, 216 is equal to or less than one-half,one-third, one-fourth, one-sixth, or one-twelfth a number of theplurality of wells 120 in the cell culture plate 114.

The methods can further include moving the sealing element 116, 216 to asecond open position, thereby bringing a second plurality of openings118, 218 in the sealing element 116, 216 into register with a secondsubset of the plurality of openings in the enclosure 102. The secondplurality of openings 118, 218 in the sealing element 116, 216 and thesecond subset of the plurality of openings in the enclosure 102, when inregister, can provide a second plurality of passages from an exterior ofthe incubator 100 to the internal chamber 110 of the enclosure 102. Insome embodiments when the sealing element 116, 216 is in the second openposition, all openings of the plurality of openings in the enclosure 102other than the second subset of openings are occluded by the sealingelement 116, 216.

The methods can further include moving the sealing element 116, 216 to athird open position, thereby bringing a third plurality of openings 118,218 in the sealing element 116, 216 into register with a third subset ofthe plurality of openings in the enclosure 102. The third plurality ofopenings 118, 218 in the sealing element 116, 216 and the third subsetof the plurality of openings in the enclosure 102, when in register, canprovide a third plurality of passages from an exterior of the incubator100 to the internal chamber 110 of the enclosure 102. In someembodiments when the sealing element 116, 216 is in the third openposition, all openings of the plurality of openings in the enclosure 102other than the third subset of openings are occluded by the sealingelement 116, 216.

Systems.

Systems are also provided including the well plate incubators 100described herein. In some embodiments, a system 200 for incubation whileproviding continuing access for import/export is provided. A schematicdiagram of one exemplary system is shown in FIG. 18. The systems 200 caninclude the well plate incubator 100, a robotic sampling component(including sampling drive controller 178 and sampling motors 182)configured to access the well plate incubator 100 to collect or depositsamples with an internal chamber of the well plate incubator, and atleast one controller. In some embodiments, the incubator 100 includescontroller 174, while in other embodiments, incubator controller 174 maybe part of the system 200. In some embodiments, sampling drivecontroller 178 and pump controller 180 may be distinct controllers ormay be part of the same controller. In any case, any of the controllers,including 174, 178 and/or 180, may be instructed by the control software176. The controller 174 may be instructed by the control software 176 toopen a plurality of passages from an exterior of the incubator 100 tothe internal chamber of the enclosure. The controller 174 may beinstructed by the control software 176 to control the robotic samplingcomponent to access, via the plurality of passages, a plurality of wells120 of a well plate 114 contained within the internal chamber 102 of theenclosure 102. The controller 174 may be instructed by the controlsoftware 176 to close the plurality of passages. The system 200 can beconfigured to maintain the internal chamber 110 of the enclosure 102under positive pressure by instructing controller 174. Additionally,sampling drive controller 178 and pump controller 180 can be configuredto control the motors 182 of the robotic sampling component and pumps184 of a hydraulic component (including pump controller 180 and pumps184) respectively of system 200, activating the import/export tip 186 towithdraw material from one of the plurality of wells 120 of the wellplate. The controllers 178 and 180 can be configured to control therobotic sampling component/hydraulic component of system 200 to deliverthe withdrawn material to another apparatus which may be external to thesystem 200. In some embodiments, the apparatus to which withdrawnmaterial is delivered may be included as an additional component ofsystem 200 and the control software 176 may instruct the additionalapparatus as well. In some embodiments, an apparatus to which materialwithdrawn from the incubator 100 by import/export tip 186 may bedelivered may be a microfluidic device 190. In some embodiments, themicrofluidic device 190 may be a nanofluidic device. In otherembodiments, material withdrawn from the incubator 100 via import/exporttip 186 may be delivered via the robotic sampling component/hydrauliccomponent of system 200 to an analytical instrument 192. Materialscultured in the system or delivered to the incubator of the system 200may be any suitable materials as described herein and may includemicro-objects and/or biological micro-objects. In some embodiments,biological micro-objects are cultured, imported and/or exported insystem 200. Some non-limiting examples of suitable analyticalinstruments to which material may be delivered include sequencinginstrumentation and sample prep therefor, assay instrumentation, massspectrometry and sample prep therefor, and storage devices andstabilization prep therefor. In yet other embodiments, materialwithdrawn from the incubator 100 via import/export tip 186 may bedelivered via the robotic sampling component to a macroscale cellculture apparatus 194.

The control software 176 can instruct the sampling drive controller 178and/or pump controller 180 to control the robotic sample componentand/or hydraulic component to deliver one or more samples of material toone or more wells 120 of the well plate contained within the well plateincubator 100. The one or more samples of material can be obtained froma macroscale cell culture apparatus 194, a microfluidic device (whichmay be a nanofluidic device) 190 or an analytical instrument 192. Themacroscale cell culture apparatus 194 may include cell culture plates,flasks or reactors. Microfluidic devices 190, which include nanofluidicdevices, include but are not limited to, droplet generation devices,microfluidic cell sorting and/or cell culturing devices. Analyticalinstruments 192 from which one or more samples of material may beobtained can include cell sorting instruments such as a flow cytometer,cell dissociation apparatuses, and cell storage apparatuses asnon-limiting examples.

The system 200 may further include a mixing tip 188, which may be partof the hydraulic component. The mixing tip 188 may also access an openedpassage to a well 120 having contents present in the cell culture plate114 to mix the contents of the well 120 prior to import and/or exportto/from the well 120. The action of mixing tip 188 may be controlled bycontroller 180. The mixing tip may rotate, vibrate, or otherwise moveabout, inject gas or liquid to effect mixing, or the like. In someembodiments, the control software 176 of system 200 may control themixing tip 188 to withdraw an aliquot of fluid from the well 120 andreinject it to mix the contents of the well 120 before material is addedto the well 120 or is withdrawn from the well 120 by the import/exporttip 186. In some embodiments, the system may control the mixing tip towithdraw about 10 μl to about 50 μl of fluid from the well and reinjectit to the well at a rate of about 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 15 μl, 17 μl, or about 20 μl/sec.In some embodiments, the mixing tip withdraws about 10 μl, 11 μl, 12 μl,13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl, 25 μl, 30 μl, 35μl, 40 μl, 45 μl, or about 50 μl of fluid to mix the contents of thewell before withdrawing from or depositing to a well 120.

The import/export tip 186 of system 200 may be connected to tubing (notshown) that can connect the incubator 100 to a microfluidic device 190,a macroscale cell culture apparatus 194, or an analytical instrument192. If connected to tubing for import and/or export of biologicalmaterial, the tubing may be made of material suitable for autoclaving ormay be disposable. The tubing typically is made of a hydrophobicmaterial. In some embodiments, the tubing may be made of Teflon™(polytetrafluoroethylene) or PEEK (polyetheretherketone). The Teflon™tubing may have a 1/16″ outer diameter, with a 1/32″ inner diameter.PEEK tubing may have a 1/32″ outer diameter with a 0.015″ innerdiameter. The latter dimensions may be used for imports/exports ofmaterial from a 384 well plate. In various embodiments, the system maycontrol the import/export tip to withdraw/deposit material at a rate ofabout 0.01 μl, 0.02 μl, 0.05 μl, 0.1 μl, 0.2 μl, 0.5 μl, 1 μl, 2 μl, 3μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 15 μl, 17μl, 20 μl, 22 μl, 24 μl, 25 μl, 27 μl, 29 μl, 30 μl per sec, or anyrange defined by two of the foregoing values.

The robotic sampling component may be selected from but not limited to alinear stages robot, an xyz robot, or a Selective ComplianceAssembly/Articulated Robot Arm (SCARA) robotic sampler. The roboticsampling component may direct the import/export tip to deposit/withdrawmaterial to/from the wells 120 of the cell culture plate 114 within theincubator 100.

EXAMPLES Example 1: CHO Cell Viability in a Well Plate Incubator

Materials:

CHO-S cells were obtained from Fisher Scientific (Invitrogen™ Freestyle™CHO-S cells, catalog #R80007). The cultures were maintained by seedingat 2×10⁵ viable cells/ml and incubating at 37° C., using 5% carbondioxide in air as the gaseous environment. Cells were split every 2-3days.

Culture Medium:

Freestyle™ Expression Medium (ThermoFisher Scientific, catalog#12651014), an animal origin-free, chemically defined, protein-freemedium, was used. It was supplemented with HT Supplement from Gibco(Cat#11067-030) and L-Glutamine 200 mM from Gibco (Cat#25030-081).

Incubator: Manufactured by Berkeley Lights, Inc., includes a lid,shutter, and temperature and environment inputs as described above.Throughout the experiment, the lid was heated to 38° C., the enclosuresurrounding the well plate was heated at 37° C., and the shutter wasclosed. Atmosphere was air supplemented with 5% CO₂, and flow rate intothe incubator was 10 L/hr for each incubator. The gas mixture washumidified to a 90% relative humidity prior to entry into the incubator.

Control Incubator:

The control incubator is commercially available (Heracel™, VIOS 160i CO₂Incubator). The control incubator was operated according to manufactureroperating directions. The atmosphere was air supplemented with 5% CO₂,and the temperature was maintained at 37° C.

Well Plates:

96-well flat-bottom well plates with low evaporation lids, non-tissueculture treated were used (Falcon, Cat#351172).

Viability Assay:

Cell Titer Glo Assay was obtained from Promega (Cat# G7572), andluminescence was measured using an EnVision Xcite Multilabel PlateReader from PerkinElmer (Cat #: 2104-002A).

For each well plate, target seeding of cells was 20 cells per well,except for H12 which was maintained empty. Loaded well plates wereplaced within the test and control incubators. A total of 4 well plateswere incubated in the test incubator and a total of 10 well plates wereincubated in the control incubator for the entire experimental period.After culturing for 24 h in the test incubator, the test incubator wellplates were transferred to the commercial incubator. All culture plateswere incubated for a further 7 days.

At the end of the eighth day after seeding, samples were taken from eachwell of each incubator plate and individually subjected to the CellTitle Glo Assay, which was performed according to manufacturerdirections. The assay generates a quantifiable fluorescent signal inproportion to ATP present, where ATP is used as a marker of cellmetabolic activity. The raw fluorescence amplitude (Data not shown) foreach well was normalized against a standard curve according to themanufacturer's directions, and the number of cells was calculatedtherefrom. From the calculated cell number, cell divisions per well werecalculated.

The number of cell divisions in each well of a well plate was graphed(shown in FIG. 24), one curve for each well plate. Data for each of thefour well plates incubated in the test incubators is represented by thecurves (heavy solid line) and data from the well plates incubatedentirely in the control incubator is represented by curves having onlyindividual closed circles (•). As presented in FIG. 24, growth rate isrepresented along the x axis, with slower growth (fewer cell divisions)on the left side of the graph and faster growth (more cell divisions) onthe right side of the graph. Values on the y axis represented individualwells for each well plate.

The curves (solid line) for well plates incubated in the test incubatorshowed no delay in growth relative to the curves for the well platesincubated entirely within the control incubator. The resultsdemonstrated a lack of deleterious effect of incubation within the testincubator during the critical initial 24 hr period after seeding.Additionally, the curves for the well plates incubated in the testincubator exhibited less spread along the x axis, which indicates moreuniform growth across the well plate (e.g., more similar number of celldivisions for all wells across the well plate) compared to the curvesrepresenting the growth of cells incubated entirely in the controlincubator.

Example 2: OKT3 Cell Viability in a Well Plate Incubator

Materials:

OKT3 cells, a murine myeloma hybridoma cell line, are obtained from theATCC (ATCC® Cat# CRL-8001™). The cells are provided as a suspension cellline. Cultures are maintained by seeding at about 1×10⁵ to about 2×10⁵viable cells/mL and incubating at 37° C., using 5% carbon dioxidegaseous environment. Cells are split every 2-3 days. OKT3 cell numberand viability are counted and cell density is adjusted to 5×10⁵/ml forloading into well plates for incubation in the continuous access wellplate incubator.

Culture Medium:

500 ml Iscove's Modified Dulbecco's Medium (IMDM, ATCC® Cat#30-2005),200 ml Fetal Bovine Serum (ATCC® Cat#30-2020) and 1 mlpenicillin-streptomycin (Life Technologies® Cat#15140-122) are combinedto make the culture medium. The complete medium is filtered through a0.22 μm filter and stored away from light at 4° C. until use. Theculture medium is conditioned with 5% carbon dioxide in air beforeintroduction into the incubator.

Incubator:

Manufactured by Berkeley Lights, Inc., includes a lid, shutter, andtemperature and environment inputs as described above. The temperatureof the incubator is maintained at 37° C. and is kept under a positivegas pressure by flowing 5% carbon dioxide in air through the incubatorat a flow rate of about 10 liters per hr. The gas mixture is humidifiedto a 90% relative humidity prior to entry into the incubator.

Cell Culture Plates:

Falcon® 96 well U bottom plates are used (Corning, Cat#351177).

Viability Assay:

Two 96 well cell culture plates are seeded, with each well receiving 10cells of OKT3 cells, 100 microliters of IMDM culture medium, prepared asdescribed above, is added to each well of both well plates. Each of thetwo well plates have the same distribution of cell types in the samelocations within the well plate. A first well plate of the two plates,is placed directly into a standard tissue culture incubator, such asHeracell™ 150i (Fisher Scientific, Cat#51026283). The secondexperimental well plate is placed into the well plate incubator havingcontinuous access. Both incubators are maintained at the sametemperature (37° C.), and under the same environmental conditions,including conditioned gas having 5% carbon dioxide. Humidity ismaintained in both systems at 90%.

After 24 h, the experimental well plate is removed from the well plateincubator having continuous access, and is put into the same model ofcommercially available tissue culture incubator as described above whichis maintained at the same conditions as described above. Both controland experimental well plates are cultured for a further 6 days. At theconclusion of 7 days total culture time, cell viability is evaluated andan approximate cell count is obtained. CellTiterGlo® (Promega Corp.)luciferase assay is used to homogeneously lyse the cells and generate aluminescent signal, oxyluciferin, proportional the amount of ATPpresent, which in turn is directly proportional to the number of cellspresent. An equivalent amount of CellTiterGlo® Reagent is added directlyto each well and the resultant luminescence is recorded on the Wallac1420 Victor²™ (PerkinElmer, Cat#1420-832). The luminescence produced isdirectly proportional to the number of viable cells, and approximatesthe number of live cells within each well. Comparative viability/growthis based on the numbers of cells/well in experimental plates vs control.

The results indicate that cell viability in the experimental well plateis at least 95% of control for each cell line evaluated.

Example 3: Culture of OKT3 Cells in a Continuous Access Well PlateIncubator and Transfer to a Microfluidic Device

Microfluidic Device Materials:

Microfluidic devices and System: Manufactured by Berkeley Lights, Inc.The system includes at least a flow controller, temperature controller,fluidic medium conditioning and pump component, light source andprojector for light activated DEP configurations, mounting stage, and acamera. The microfluidic device includes flow channels and pens for cellisolation, assay, and/or growth, with single pen volume of approximately1.5×10⁶ μm³.

Transfer Component of the System:

A linear stages robot, import/export tip having an outer diameter of1.067 mm of the microfluidic device.

Priming Solution for Microfluidic Device:

The culture medium (as described in Example 2) containing 0.1% Pluronic®F127 (Life Technologies® Cat# P6866).

Preparation of the Microfluidic Device Prior to Transfer:

The microfluidic device is loaded onto the system and purged with 100%carbon dioxide at 15 psi for 5 min. Immediately following the carbondioxide purge, the priming solution is perfused through the microfluidicdevice at 8 μl/sec until a total volume of 2.5 ml is perfused throughthe microfluidic device. The culture medium is then flowed through themicrofluidic device at 8 μl/sec until a total of 1 ml of culture mediumis perfused through the microfluidic device. The temperature of themicrofluidic device is maintained at 37° C. Culture medium is perfusedthroughout the experiment using a variable perfusion method whichincludes one 4 h period of perfusion at 0.01 μl/sec, followed by a shorthigh velocity perfusion at 8 μl/sec for about 3 sec, interspersed byshort perfusion stop periods of less than a minute.

Experiment:

OKT3 cells are seeded into each well of a 96 well cell culture plate.The cells are cultured within the continuous access well plate incubatorfor 1 day. At the end of the culture period, analysis to determine cellviability and numbers of cells is performed on one well of the 96 wellplate. After determining that viability and growth requirements havebeen met, the microfluidic device is prepared for transfer. The openingsin the enclosure of the well plate incubator are opened by thecontroller of the well plate incubator, and positive gas flow iscontinued. For each well transferred, a mixing tip is first introducedthrough the opening in the enclosure connecting the well to the exteriorenvironment. Agitation is provided by withdrawing and then re-injecting50 μl of the culture medium within the well to mix the cells,additionally dislodging any cells adhering to the walls of the well.Agitation is performed either prior to inserting the import/export tipor at the same time. A sample of the contents of the well is drawn intothe import/export tip and delivered to the input of the microfluidicdevice. The cells are moved via flow, gravity or by dielectrophoresisforces through the channel of the microfluidic device and then placedinto individual pens of the microfluidic device for further evaluation.

Numbered Embodiments of the Invention

1. An incubator comprising: an enclosure having an internal chamberconfigured to support a cell culture plate comprising a plurality ofwells, wherein the enclosure comprises a plurality of openingsconfigured to allow access to the wells of the cell culture plate; atemperature controller configured to maintain a temperature of theinternal chamber within a desired range; a first heating/cooling deviceengaged directly or indirectly with the enclosure, the firstheating/cooling device controlled by the temperature controller; and asealing element comprising a first plurality of openings correspondingto at least a subset of the plurality of openings in the enclosure,wherein the sealing element is movable between a closed position wherethe sealing element occludes, and thereby seals, each of the pluralityof openings in the enclosure and a first open position where the firstplurality of openings of the sealing element are in register with the atleast a subset of the plurality of openings in the enclosure, therebyproviding access to the internal chamber of the enclosure and any cellculture plate contained therein.

2. The incubator according to embodiment 1, further comprising: at leastone passage in the enclosure configured for gas entry; and a connectoradapted to connect a pressurized gas source to the at least one passage,wherein the sealing element is configured to form a seal with theplurality of openings in the enclosure that allows the enclosure tomaintain a pressure in the internal chamber between about 0.0005 psi toabout 0.01000 psi above ambient pressure when gas from the pressurizedgas source flows into the internal chamber.

3. The incubator according to any one of embodiments 1-2, wherein eachopening of the plurality of openings in the enclosure has a diameter ofabout 1 mm to about 10 mm or about 1.0 mm, about 1.5 mm, about 2.0 mm,about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm,about 5.0 mm, about 5.5 mm, about 6.0 mm, about 6.5 mm, about 7.0 mm,about 7.5 mm, about 8.0 mm, about 8.5 mm, about 9.0 mm, about 9.5 mm, orabout 10.0 mm or any range defined by one of the foregoing sizes.

4. The incubator according to any previous embodiment, wherein theinternal chamber has a volume of about 200 cm³ to about 750 cm³.

5. The incubator according to any previous embodiment, wherein theinternal chamber has a volume of about 750 cm³ to about 2000 cm³.

6. The incubator according to any previous embodiment, wherein the cellculture plate is a 96-well plate or a 384-well plate.

7. The incubator according to any previous embodiment, wherein theplurality of openings in the enclosure are configured to be in registerwith the plurality of wells in the cell culture plate.

8. The incubator according to any previous embodiment, wherein theinternal chamber includes a reservoir configured to hold a fluid.

9. The incubator according to any previous embodiment, wherein theenclosure comprises a base and a lid, the base and the lid defining theinternal chamber.

10. The incubator according to any previous embodiment, wherein theenclosure comprises a base, a lid, and a front plate, the base, the lidand the front plate defining the internal chamber.

11. The incubator according to any one of embodiments 9-10, wherein thebase is formed from a rigid material having a high thermal conductivityand low thermal capacitance.

12. The incubator according to any one of embodiments 9-11, wherein thebase is configured with a hollow region forming part or all of theinternal chamber of the enclosure.

13. The incubator according to any one of embodiments 10-12, wherein thebase includes a bottom and four walls with one of the four walls havinga height that is shorter than the height of the other three walls.

14. The incubator according to any one of embodiments 9-13, wherein thelid is formed from an insulating plastic.

15. The incubator according to any one of embodiments 9-14, wherein thelid includes an outer surface and an inner surface within the enclosure,the inner surface including one or more recesses.

16. The incubator according to embodiment 15, wherein the lid furtherincludes an adhesive layer adhered to the inner surface, and wherein theadhesive layer is configured to prevent air within the enclosure fromfilling the one or more recesses.

17. The incubator according to any one of embodiments 15 or 16, whereinthe one or more recesses substantially surround groups of openings ofthe plurality of openings in the enclosure, each group comprising two ormore openings of the plurality.

18. The incubator according to any one of embodiments 9-17, wherein thelid includes one or more connectors configured to sealably connect thelid to the base.

19. The incubator according to embodiment 18, wherein the one or moreconnectors include a magnet, a flexible tab, and/or a clip.

20. The incubator according to embodiment 18, wherein the one or moreconnectors are flexible tabs, and wherein each flexible tab isconfigured to engage with a pin and thereby secure the lid to the base.

21. The incubator according to any previous embodiment, wherein thefirst plurality of openings of the sealing element is the same as thenumber of openings in the enclosure.

22. The incubator according to any previous embodiment, wherein thesealing element further comprises a second plurality of openings, thesecond plurality of openings being different from the first plurality ofopenings.

23. The incubator according to embodiment 22, wherein the number ofopenings in the first plurality of openings and/or the second pluralityof openings in the sealing element is less than the number of openingsin the enclosure.

24. The incubator according to any one of embodiments 22-23, wherein thenumber of openings in the second plurality of openings in the sealingelement is one-half, one-third, or one-fourth the number of openings inthe enclosure.

25. The incubator according to any previous embodiment, wherein thesealing element is movable between a closed position, a first openposition, and a second open position, and wherein: when the sealingelement is in the closed position, each of the plurality of the openingsin the enclosure are occluded; when the sealing element is in the firstopen position, the first plurality of openings in the sealing elementare in register with a first subset of the plurality of opening in theenclosure and all other openings of the plurality of openings in theenclosure are occluded; and when the sealing element is in the secondopen position, the first plurality of openings in the sealing elementare in register with a second subset of openings in the enclosure andall other openings of the plurality of openings in the enclosure areoccluded.

26. The incubator according to embodiment 25, wherein the first subsetof openings in the enclosure and the second subset of openings in theenclosure are non-overlapping subsets.

27. The incubator according to any previous embodiment, wherein thesealing element is located inside the internal chamber of the enclosure.

28. The incubator according to any previous embodiment, furthercomprising: a sealing element actuator configured to move the sealingelement between a first open position and a closed position.

29. The incubator according to embodiment 28, wherein the sealingelement actuator is configured to move the sealing element between asecond open position and the closed position or the first open position.

30. The incubator according to embodiment 29, wherein moving the sealingelement to the second open position includes lining up the firstplurality of openings of the sealing element with a second subset of theplurality of openings in the enclosure, wherein the second subset of theplurality of the openings in the enclosure is less than the plurality ofopenings in the enclosure.

31. The incubator according to any one of embodiments 28-30, wherein thesealing element actuator comprises a motor or rotary solenoid.

32. The incubator according to any previous embodiment, furthercomprising a printed circuit board (PCB).

33. The incubator according to embodiment 32, wherein the enclosureincludes a base and a lid and the sealing element is located between thePCB and the lid.

34. The incubator according to any one of embodiments 32-33, furthercomprising: one or more sensors on the PCB.

35. The incubator according to embodiment 34, wherein each of the one ormore sensors is selected from the group consisting of: a temperaturesensor, a humidity sensor, an oxygen sensor, and a carbon dioxidesensor.

36. The incubator according to any previous embodiment, wherein thefirst heating/cooling device is selected from the group consisting of: aresistive heater, a fluid coil configured to circulate a heat exchangefluid, one or more Peltier devices, and combinations thereof.

37. The incubator according to any previous embodiment, wherein thefirst heating/cooling device directly contacts or indirectly providesheat transfer to an outer surface of the bottom of the enclosure.

38. The incubator according to embodiment 37, wherein the firstheating/cooling device contacts at least about 75% of the outer surfaceof the bottom of the enclosure.

39. The incubator according to any previous embodiment, wherein thefirst heating/cooling device comprises a fluid coil.

40. The incubator according to any previous embodiment, furthercomprising: a second heating/cooling device, wherein the secondheating/cooling device is adjacent a top of the enclosure and iscontrolled by the temperature controller.

41. The incubator according to embodiment 40, wherein the secondheating/cooling device is within the enclosure.

42. The incubator according to any one of embodiments 40-41, wherein thesecond heating/cooling device comprises a plurality of openings that arein register with the plurality of openings in the enclosure.

43. The incubator according to any one of embodiments 40-42, wherein thesecond heating/cooling device comprises resistive heating elements thatare part of the PCB.

44. The incubator according to embodiment 43, wherein the PCB comprisesa plurality of openings in register with the plurality of openingspassing through the enclosure.

45. The incubator according to any one of embodiments 43-44, wherein theresistive heating elements are positioned internally to the PCB as partof a multi-layer construction of the PCB.

46. The incubator of any one of the preceding embodiments, furthercomprising: a support for the cell culture plate.

47. The incubator according to embodiment 46, wherein the support isconfigured to slideably move relative to the enclosure from a positionwithin the enclosure to a position outside of the internal chamber ofthe enclosure.

48. The incubator according to any one of embodiments 46-47, furthercomprising a distal lip on the support configured to engage an edge ofthe cell culture plate.

49. The incubator according to any one of embodiments 46-48, furthercomprising: an access door engaged with the support for the cell cultureplate.

50. The incubator according to embodiment 49, wherein the support andaccess door form an access assembly including a front plate thatsealably interfaces with a portion of the enclosure.

51. The incubator according to embodiment 50, further comprising: abiased connection between the front plate and the access door configuredto provide a compressive force to the front plate.

52. The incubator according to any one of embodiments 50-51, wherein theaccess assembly is movably mounted on an enclosure support that supportsthe enclosure.

53. The incubator according to embodiment 52, further comprising: trackson the enclosure support, wherein the access assembly is configured toslide relative to the tracks on the enclosure support.

54. The incubator according to embodiment 53, the access assemblyfurther comprising rails configured to slide relative to the tracks onthe enclosure support.

55. The incubator according to embodiment 54, further comprising: anengagement surface on the rails configured to engage with acomplementary structure of the enclosure support to secure a position ofthe access assembly relative to the enclosure support.

56. The incubator according to embodiment 55, wherein the position ofthe access assembly corresponds to an open or closed position of theaccess assembly.

57. The incubator according to any one of embodiments 50-56, furthercomprising: a door switch configured to mechanically, electronically, ormagnetically engage with a complementary structure of the accessassembly.

58. The incubator according to embodiment 46, wherein the support isformed by one or more internal surfaces of the enclosure.

59. The incubator according to embodiment 2, wherein the at least onepassage configured for gas entry is located on a wall of the base at thesame height from a bottom of the base as a side of the cell cultureplate.

60. The incubator according to any previous embodiment, furthercomprising: at least one fluid drain passage in the enclosure configuredto drain a fluid reservoir within the enclosure, wherein the fluid drainpassage is sealable.

61. The incubator according to any previous embodiment, furthercomprising: an insulation material coupled to the enclosure.

62. The incubator according to embodiment 61, wherein the insulationmaterial is attached to one or more outer surfaces of the enclosure.

63. The incubator according to any previous embodiment, wherein theincubator is configured to maintain a selected internal temperature,humidity, and gas content within the internal chamber of the enclosure.

64. The incubator according to embodiment 63, further comprising: acontroller configured to maintain the selected internal temperature,humidity, and gas content within the internal chamber of the enclosure.

65. The incubator according to any previous embodiment, furthercomprising: an enclosure support configured to support the enclosure.

66. The incubator according to embodiment 65, further comprising: one ormore adjustable connectors configured to connect the enclosure supportto the enclosure.

67. The incubator according to any previous embodiment, wherein eachopening of the plurality of openings in the enclosure has a diameter ofabout 1 mm to about 5 mm or about 1.0 mm, about 1.5 mm, about 2.0 mm,about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, orabout 5.0 mm or any range defined by one of the foregoing sizes.

68. The incubator according to any previous embodiment, wherein each ofthe plurality of openings in the sealing element has a diameter of about1 mm to about 10 mm or about 1.0 mm, about 1.5 mm, about 2.0 mm, about2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, about5.0 mm, about 5.5 mm, about 6.0 mm, about 6.5 mm, about 7.0 mm, about7.5 mm, about 8.0 mm, about 8.5 mm, about 9.0 mm, about 9.5 mm, or about10.0 mm or any range defined by one of the foregoing sizes.

69. The incubator according to any previous embodiment, wherein each ofthe plurality of openings in the sealing element has a diameter of about1 mm to about 5 mm or about 1.0 mm, about 1.5 mm, about 2.0 mm, about2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, or about5.0 mm, or any range defined by one of the foregoing sizes.

70. A method for accessing an internal chamber of an incubator, whereinthe incubator comprises an enclosure having a plurality of openings anda sealing element having a plurality of openings corresponding to atleast a subset of the plurality of openings in the enclosure, the methodcomprising: moving the sealing element to an open position and therebybringing the plurality of openings in the sealing element into registerwith a first subset of openings of the plurality of openings in theenclosure, the plurality of openings in the sealing element and thefirst subset of openings of the plurality of openings in the enclosurethereby providing a first plurality of passages from an exterior of theincubator to an internal chamber of the enclosure; advancing animport/export tip through one or more of the first plurality of passagesbetween the exterior of the incubator and the internal chamber of theenclosure; and collecting or depositing a material within the internalchamber of the enclosure via the import/export tip.

71. The method according to embodiment 70, wherein collecting ordepositing the material comprises collecting or depositing the materialwithin a well of a cell culture plate within the internal chamber of theenclosure.

72. The method according to any one of embodiments 70-71, furthercomprising: withdrawing the import/export tip through one or more of thepassages between the exterior of the incubator and the internal chamberof the enclosure after collecting or depositing the material.

73. The method according to embodiment 72, further comprising: movingthe sealing element to a closed position such that the sealing elementoccludes the plurality of openings in the enclosure.

74. The method according to any one of embodiments 70-73, wherein thesealing element is in the open position for an amount of time which issufficiently short so as to prevent a carbon dioxide content and/or ahumidity of air present in the internal chamber of the incubator fromequilibrating with a carbon dioxide content and/or a humidity of airsurrounding the incubator.

75. The method according to any one of embodiments 70-74, comprising:actuating a sealing element actuator to move the sealing element to theopen position or closed position.

76. The method according to embodiment 75, wherein actuating the sealingelement actuator comprises activating a motor or rotary solenoid.

77. The method according to any one of embodiments 70-76, wherein movingthe sealing element between the open position and closed positioncomprises sliding the sealing element relative to the enclosure.

78. The method according to any one of embodiments 70-77, wherein whenthe plurality of openings in the sealing element are in the openposition, the plurality of openings are configured to be in registerwith a plurality of wells in the cell culture plate.

79. The method according to any one of embodiments 70-78, wherein theincubator comprises a support within the internal chamber of theenclosure configured to support the cell culture plate.

80. The method according to embodiment 79, further comprising: slidingthe support to a position outside of the internal chamber of theenclosure and thereby withdrawing the cell culture plate from theinternal chamber of the enclosure.

81. The method according to embodiment 80, wherein sliding the supportincludes sliding an access door attached to the support.

82. The method according to any one of embodiments 80-81, whereinsliding the support includes sliding along one or more tracks of anenclosure support of the incubator.

83. The method according to any one of embodiments 80-82, whereinsliding the support is performed by a human operator.

84. The method according to any one of embodiments 80-82, whereinsliding the support is performed robotically.

85. The method according to embodiment 79, further comprising: slidingthe support to withdraw the support from the internal chamber of theincubator to a position outside of the internal chamber of theenclosure.

86. The method according to embodiment 85, further comprising: placing acell culture plate on the support while the support is in the positionoutside of the internal chamber of enclosure.

87. The method according to embodiment 86, wherein placing the cellculture plate is performed by a human operator.

88. The method according to embodiment 86, wherein placing the cellculture plate is performed robotically.

89. The method according to any one of embodiments 85-88, furthercomprising: sliding the support to a position inside the internalchamber of the enclosure and thereby moving the cell culture plate intothe internal chamber of the enclosure.

90. The method according to embodiment 89, wherein sliding the supportcomprises sliding an access door attached to the support for the cellculture plate.

91. The method according to any one of embodiments 89 or 90, whereinsliding the support comprises sliding along one or more tracks of anenclosure support of the incubator.

92. The method according to any one of embodiments 89-91, whereinsliding the support is performed by a human operator.

93. The method according to any one of embodiments 89-91, whereinsliding the support is performed robotically.

94. The method according to any one of embodiments 70-93, furthercomprising: measuring one or more of a temperature, a humidity, and acarbon dioxide content of the internal chamber of the enclosure.

95. The method according to any one of embodiments 70-94, furthercomprising: controlling one or more of a temperature, a humidity, and acarbon dioxide content of the internal chamber of the enclosure.

96. The method according to embodiment 95, wherein controlling thetemperature comprises heating or cooling the internal chamber of theenclosure.

97. The method according to any one of embodiments 95 or 96, whereincontrolling the humidity comprises providing a humidity source to theinternal chamber of the enclosure.

98. The method according to any one of embodiments 95-97, whereincontrolling the carbon dioxide content comprises providing a gas sourcecomprising carbon dioxide to the internal chamber of the incubator.

99. The method according to embodiment 98, wherein the gas sourcecomprising carbon dioxide further comprises oxygen and nitrogen.

100. The method according to any one of embodiments 70-99, wherein thesealing element, when in the closed position, is capable of maintaininga pressure within the internal chamber of the enclosure between about0.0005 psi to about 0.0100 psi above ambient pressure.

101. The method according to embodiment 100, further comprising:providing a purge gas to the internal chamber of the enclosure whereby,when the sealing element is in the closed position and the support forthe cell culture plate is positioned inside the internal chamber of theenclosure, the pressure within the internal chamber of the enclosure ismaintained between about 0.0005 psi to about 0.0100 psi above ambientpressure.

102. The method according to any one of embodiments 70-101, wherein theimport/export tip comprises a plurality of tips.

103. The method according to embodiment 102, further comprising:simultaneously collecting or depositing the material from a plurality ofthe wells of the cell culture plate using the plurality of tips of theimport/export tip.

104. The method according to embodiment 103, wherein collecting ordepositing the material is performed robotically.

105. The method according to any one of embodiments 70-104, furthercomprising: maintaining a pressure within the internal chamber of theincubator that is greater than a pressure outside of the incubator whenthe sealing element is in an open position.

106. The method according to any one of embodiments 70-105, wherein eachopening of the plurality of openings in the enclosure has a diameter ofabout 1 mm to about 10 mm or about 1.0 mm, about 1.5 mm, about 2.0 mm,about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm,about 5.0 mm, about 5.5 mm, about 6.0 mm, about 6.5 mm, about 7.0 mm,about 7.5 mm, about 8.0 mm, about 8.5 mm, about 9.0 mm, about 9.5 mm, orabout 10.0 mm or any range defined by one of the foregoing sizes.

107. The method according to any one of embodiments 70-105, wherein eachopening of the plurality of openings in the enclosure has a diameter ofabout 1 mm to about 5 mm, or about 1.0 mm, about 1.5 mm, about 2.0 mm,about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm, about 4.5 mm, orabout 5.0 mm, or any range defined by one of the foregoing sizes.

108. The method according to any one of embodiments 70-107, wherein eachopening of the plurality of openings in the sealing element has adiameter of about 1 mm to about 10 mm, or about 1.0 mm, about 1.5 mm,about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm,about 4.5 mm, about 5.0 mm, about 5.5 mm, about 6.0 mm, about 6.5 mm,about 7.0 mm, about 7.5 mm, about 8.0 mm, about 8.5 mm, about 9.0 mm,about 9.5 mm, or about 10.0 mm or any range defined by one of theforegoing sizes.

109. The method according to any one of embodiments 70-107, wherein eachopening of the plurality of openings in the sealing element has adiameter of about 1 mm to about 5 mm, or about 1.0 mm, about 1.5 mm,about 2.0 mm, about 2.5 mm, about 3.0 mm, about 3.5 mm, about 4.0 mm,about 4.5 mm, or about 5.0 mm, or any range defined by one of theforegoing sizes.

110. The method according to any one of embodiments 70-109, wherein thematerial collected or deposited in the internal chamber of the enclosurecomprises a biological micro-object.

111. The method according to any one of embodiments 70-110, furthercomprising: establishing an environment within the internal chamber ofthe enclosure to support a biological micro-object cultured in the cellculture plate.

112. A method for accessing an internal chamber of an incubator, whereinthe incubator comprises an enclosure having a plurality of openings anda sealing element having more than one plurality of openings, whereineach plurality of openings in the sealing element corresponds to atleast a subset of the plurality of openings in the enclosure, the methodcomprising: moving the sealing element to a first open position andthereby bringing a first plurality of openings in the sealing elementinto register with a first subset of the plurality of openings in theenclosure, the first plurality of openings in the sealing element andthe first subset of openings in the plurality of openings in theenclosure providing, when in register, a first plurality of passagesfrom an exterior of the incubator to the internal chamber of theenclosure; advancing an import/export tip through one or more of thefirst plurality of passages between the exterior of the incubator andthe internal chamber of the enclosure; and collecting or depositing amaterial with the import/export tip within the internal chamber of theenclosure.

113. The method according to embodiment 112, wherein, when the sealingelement is in the open position, the first plurality of openings in thesealing element are configured to be in register with a first subset ofa plurality of wells in the cell culture plate.

114. The method according to any one of embodiments 112 or 113, whereina number of the plurality of openings in the sealing element is the sameas a number of the plurality of wells in the cell culture plate.

115. The method according to any one of embodiments 112 or 113, whereina number of the plurality of openings in the sealing element is equal toor less than one-half, one-third, one-fourth, one-sixth, or one-twelftha number of the plurality of wells in the cell culture plate.

116. The method according to any one of embodiments 112-115, furthercomprising: moving the sealing element to a second open position,thereby bringing a second plurality of openings in the sealing elementinto register with a second subset of the plurality of openings in theenclosure, the second plurality of openings in the sealing element andthe second subset of the plurality of openings in the enclosure, when inregister, providing a second plurality of passages from an exterior ofthe incubator to the internal chamber of the enclosure.

117. The method according to embodiment 116, wherein, when the sealingelement is in the second open position, all openings of the plurality ofopenings in the enclosure other than the second subset of openings areoccluded by the sealing element

118. The method according to any one of embodiments 112-117, furthercomprising: moving the sealing element to a third open position, therebybringing a third plurality of openings in the sealing element intoregister with a third subset of the plurality of openings in theenclosure, the third plurality of openings in the sealing element andthe third subset of the plurality of openings in the enclosure, when inregister, providing a third plurality of passages from an exterior ofthe incubator to the internal chamber of the enclosure.

119. The method according to embodiment 118, wherein, when the sealingelement is in the third open position, all openings of the plurality ofopenings in the enclosure other than the third subset of openings areoccluded by the sealing element.

120. The method according to any one of embodiments 112-119, furthercomprising: moving the sealing element to a closed position, and therebyoccluding each of the plurality of the openings in the enclosure.

121. A system for incubation, comprising: the well plate incubatoraccording to any one of embodiments 1-69; a robotic sampling componentconfigured to access the well plate incubator to collect or depositsamples within an internal chamber of an enclosure of the well plateincubator; and at least one controller configured to: open a pluralityof passages from an exterior of the incubator to the internal chamber ofthe enclosure; and control the robotic sampling component to access, viathe plurality of passages, a plurality of wells of a well platecontained within the internal chamber of the enclosure.

122. The system according to embodiment 121, wherein the at least onecontroller is further configured to close the plurality of passages.

123. The system according to any one of embodiments 121-122, wherein thesystem is configured to maintain the internal chamber of the enclosureunder positive pressure.

124. The system according to any one of embodiments 121-123, wherein theat least one controller is configured to control the robotic samplingcomponent to withdraw a material from one of the plurality of wells ofthe well plate.

125. The system according to embodiment 124, wherein the at least onecontroller is configured to control the robotic sample component todeliver the withdrawn material to a microfluidic device.

126. The system according to embodiment 124, wherein the at least onecontroller is configured to control the robotic sample component todeliver the withdrawn material to an analytical instrument.

127. The system according to any one of embodiments 121-126, wherein thematerial comprises a biological micro-object.

128. The system according to any one of embodiments 121-127, wherein theat least one controller is configured to control the robotic samplingcomponent to deliver one or more materials to one or more wells of thewell plate contained within the well plate incubator.

129. The system according to embodiment 128, wherein the one or morematerials are obtained from a microfluidic device.

130. The system according to embodiment 128, wherein the one or morematerials are obtained from an analytical instrument.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims. Any titles or subdivisions within the description are meantfor ease of reading and is in no way intended to limit the invention andthe combinations and sub-combinations described herein.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

1. An incubator comprising: an enclosure having an internal chamberconfigured to support a cell culture plate comprising a plurality ofwells, wherein the enclosure comprises at least one passage configuredfor gas entry, a connector adapted to connect a pressurized as source tothe at least one passage, and a plurality of openings configured toallow access to the wells of the cell culture plate; a controllerconfigured to maintain a temperature of the internal chamber within adesired range; a first heating/cooling device engaged directly orindirectly with the enclosure, the first heating/cooling devicecontrolled by the temperature controller; and a sealing elementcomprising a first plurality of openings corresponding to at least asubset of the plurality of openings in the enclosure, wherein thesealing element is configured to form a seal with the plurality ofopenings in the enclosure that allows the enclosure to maintain apressure in the internal chamber between about 0.0005 psi to about0.01000 psi above ambient pressure when gas from the pressurized gassource flows into the internal chamber, wherein the sealing element ismovable between a closed position where the sealing element occludes,and thereby seals, each of the plurality of openings in the enclosureand a first open position where the first plurality of openings of thesealing element are in register with the at least a subset of theplurality of openings in the enclosure, thereby providing access to theinternal chamber of the enclosure and any cell culture plate containedtherein, and wherein the incubator is configured to maintain a selectedinternal temperature, humidity, and gas content within the internalchamber of the enclosure.
 2. (canceled)
 3. The incubator of claim 1,wherein each opening of the plurality of openings in the enclosure has adiameter of about 1 mm to about 10 mm, or about 1 mm to about 5 mm. 4.The incubator of claim 1, wherein the internal chamber has a volume ofabout 200 cm³ to about 750 cm³ or about 750 cm³ to about 2000 cm³. 5.(canceled)
 6. The incubator of claim 1, wherein the cell culture plateis a 96-well plate or a 384-well plate.
 7. The incubator of claim 1,wherein the plurality of openings in the enclosure are configured to bein register with the plurality of wells in the cell culture plate. 8.(canceled)
 9. The incubator of claim 1, wherein the enclosure comprisesa base and a lid, the base and the lid defining the internal chamber.10. The incubator of claim 1, wherein the enclosure comprises a base, alid, and a front plate, the base, the lid and the front plate definingthe internal chamber.
 11. The incubator of claim 9, wherein the base isformed from a rigid material having a high thermal conductivity and lowthermal capacitance and is configured with a hollow region forming partor all of the internal chamber of the enclosure, and wherein the lid isformed from an insulating plastic.
 12. (canceled)
 13. The incubator ofclaim 10, wherein the base is formed from a rigid material having a highthermal conductivity and low thermal capacitance, wherein the baseincludes a bottom and four walls with one of the four walls having aheight that is shorter than the height of the other three walls and isconfigured with a hollow region forming part or all of the internalchamber of the enclosure, and wherein the base is formed from a rigidmaterial having a high thermal conductivity and low thermal capacitance.14.-17. (canceled)
 18. The incubator of claim 9, wherein the lidincludes one or more connectors configured to sealably connect the lidto the base, and wherein each of the one or more connectors are selectedfrom the group consisting of a magnet, a flexible tab, and/or a clip.19. The incubator of claim 10, wherein the lid includes one or moreconnectors configured to sealably connect the lid to the base, andwherein each of the one or more connectors are selected from the groupconsisting of a magnet, a flexible tab, and/or a clip. 20.-24.(canceled)
 25. The incubator of claim 1, wherein the sealing element ismovable between a closed position, a first open position, and a secondopen position, and wherein: when the sealing element is in the closedposition, each of the plurality of the openings in the enclosure areoccluded; when the sealing element is in the first open position, thefirst plurality of openings in the sealing element are in register witha first subset of the plurality of opening in the enclosure and allother openings of the plurality of openings in the enclosure areoccluded; and when the sealing element is in the second open position,the first plurality of openings in the sealing element are in registerwith a second subset of openings in the enclosure and all other openingsof the plurality of openings in the enclosure are occluded.
 26. Theincubator of claim 25, wherein the first subset of openings in theenclosure and the second subset of openings in the enclosure arenon-overlapping subsets.
 27. The incubator of claim 1, wherein thesealing element is located inside the internal chamber of the enclosure.28. The incubator of claim 25 further comprising: a sealing elementactuator configured to move the sealing element between the first openposition, the second open position, and the closed position. 29.-32.(canceled)
 33. The incubator of claim 43, wherein the enclosure includesa base and a lid, and wherein the sealing element is located between thePCB and the lid.
 34. (canceled)
 35. The incubator of claim 40, whereineach of the one or more sensors is selected from the group consistingof: a temperature sensor, a humidity sensor, an oxygen sensor, and acarbon dioxide sensor.
 36. The incubator of claim 1, wherein the firstheating/cooling device is selected from the group consisting of: aresistive heater, a fluid coil configured to circulate a heat exchangefluid, one or more Peltier devices, and combinations thereof. 37.-39.(canceled)
 40. The incubator of claim 1 further comprising: a secondheating/cooling device, wherein the second heating/cooling device iswithin the enclosure and is controlled by the temperature controller.41.-42. (canceled)
 43. The incubator of claim 40, wherein the secondheating/cooling device comprises a PCB having resistive heating elementsand one or more sensors, and wherein the PCB comprises a plurality ofopenings in register with the plurality of openings passing through theenclosure. 44.-45. (canceled)
 46. The incubator of claim 1 furthercomprising: a support for the cell culture plate.
 47. The incubator ofclaim 46, wherein the support is configured to slideably move relativeto the enclosure from a position within the enclosure to a positionoutside of the internal chamber of the enclosure.
 48. (canceled)
 49. Theincubator of claim 46 further comprising: an access door engaged withthe support for the cell culture plate, wherein the support and accessdoor form an access assembly including a front plate that sealablyinterfaces with a portion of the enclosure.
 50. (canceled)
 51. Theincubator of claim 49 further comprising: a biased connection betweenthe front plate and the access door configured to provide a compressiveforce to the front plate.
 52. The incubator of claim 49 furthercomprising: an enclosure support configured to support the enclosure,wherein the access assembly is movably mounted on the enclosure support.53. The incubator of claim 52, wherein: the enclosure support comprisestracks; the access assembly comprises rails configured to slide relativeto the tracks on the enclosure support, the rails having an engagementsurface configured to engage with a complementary structure of theenclosure support to secure a position of the access assembly relativeto the enclosure support, and the secured position of the accessassembly corresponds to an open or closed position of the accessassembly. 54.-57. (canceled)
 58. The incubator of claim 46, wherein thesupport is formed by one or more internal surfaces of the enclosure.59.-63. (canceled)
 64. The incubator of claim 1, wherein the controlleris further configured to maintain the humidity and gas content withinthe internal chamber of the enclosure. 65.-67. (canceled)
 68. Theincubator of claim 1, wherein each of the plurality of openings in thesealing element has a diameter of about 1 mm to about 10 mm, or about 1mm to about 5 mm.
 69. (canceled)
 70. A method for accessing an internalchamber of an incubator, wherein the incubator comprises an enclosurehaving a plurality of openings and a sealing element having a pluralityof openings corresponding to at least a subset of the plurality ofopenings in the enclosure, the method comprising: moving the sealingelement to an open position, thereby bringing the plurality of openingsin the sealing element into register with a first subset of openings ofthe plurality of openings in the enclosure, the plurality of openings inthe sealing element and the first subset of openings of the plurality ofopenings in the enclosure thereby providing a first plurality ofpassages from an exterior of the incubator to an internal chamber of theenclosure; advancing an import/export tip through one or more of theplurality of passages between the exterior of the incubator and theinternal chamber of the enclosure; and collecting or depositing amaterial within the internal chamber of the enclosure via theimport/export tip; withdrawing the import/export tip through one or moreof the passages between the exterior of the incubator and the internalchamber of the enclosure after collecting or depositing the material;and moving the sealing element to a closed position such that thesealing element occludes the plurality of openings in the enclosure,wherein the sealing element is in the open position for an amount oftime which is sufficiently short so as to prevent a carbon dioxidecontent and/or a humidity of air present in the internal chamber of theincubator from equilibrating with a carbon dioxide content and/or ahumidity of air surrounding the incubator.
 71. The method of claim 70,wherein collecting or depositing the material comprises collecting ordepositing the material within a well of a cell culture plate within theinternal chamber of the enclosure. 72.-111. (canceled)
 112. A method foraccessing an internal chamber of an incubator, wherein the incubatorcomprises an enclosure having a plurality of openings and a sealingelement having more than one plurality of openings, wherein eachplurality of openings in the sealing element corresponds to at least asubset of the plurality of openings in the enclosure, the methodcomprising: moving the sealing element to a first open position andthereby bringing a first plurality of openings in the sealing elementinto register with a first subset of the plurality of openings in theenclosure, the first plurality of openings in the sealing element andthe first subset of openings in the plurality of openings in theenclosure providing, when in register, a first plurality of passagesfrom an exterior of the incubator to the internal chamber of theenclosure; advancing an import/export tip through one or more of thefirst plurality of passages between the exterior of the incubator andthe internal chamber of the enclosure; and collecting or depositing amaterial with the import/export tip within the internal chamber of theenclosure. 113.-120. (canceled)
 121. A system for incubation,comprising: the well plate incubator of claim 1; a robotic samplingcomponent configured to access the well plate incubator to collect ordeposit samples within an internal chamber of an enclosure of the wellplate incubator; and at least one controller configured to: open aplurality of passages from an exterior of the incubator to the internalchamber of the enclosure; and control the robotic sampling component toaccess, via the plurality of passages, a plurality of wells of a wellplate contained within the internal chamber of the enclosure. 122.-130.(canceled)